Methods for treating hair loss disorders

ABSTRACT

The invention provides for methods for treating a hair loss disorder in a subject by administering a Janus Kinase/Signal Transducers and Activators of Transcription inhibitor.

This application is a continuation of U.S. patent application Ser. No.15/668,814, filed Aug. 4, 2017; which is a continuation of U.S. patentapplication Ser. No. 14/821,623, filed Aug. 7, 2015; which is acontinuation of U.S. patent application Ser. No. 13/886,252, filed May2, 2013, now U.S. Pat. No. 9,198,911; which is a continuation-in-part ofPCT Application No. PCT/US2011/59029, filed Nov. 2, 2011, which claimsthe benefit of and priority to U.S. Provisional Application No.61/409,509, filed Nov. 2, 2010, and this application is also acontinuation-in-part of PCT Application No. PCT/US2013/34688, filed Mar.29, 2013, which claims the benefit of and priority to U.S. ProvisionalPatent Application No. 61/617,225, filed on Mar. 29, 2012, and U.S.Provisional Patent Application No. 61/645,499, filed on May 10, 2012,and the contents of each of the above-listed applications are herebyincorporated by reference in their entireties.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

GOVERNMENT INTERESTS

This invention was made with government support under AR056016 andAR061881 awarded by the National Institutes of Health The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Alopecia Areata (AA) is one of the most highly prevalent autoimmunediseases affecting over 5 million individuals in the US, and as many as140 million worldwide AA leads to hair loss due to the collapse ofimmune privilege of the hair follicle and subsequent autoimmunedestruction. AA is a skin disease which leads to hair loss on the scalpand elsewhere. In some severe cases, it can progress to complete loss ofhair on the head or body. Although Alopecia Areata is believed to becaused by autoimmunity, the gene level diagnosis and rationally targetedtherapeutics have not been developed. The genetic basis of AA is largelyunknown. Its psychological impact on affected patients is devastating,particularly in children.

SUMMARY OF THE INVENTION

An aspect of the invention encompasses a method of treating a hair-lossdisorder in a mammalian subject in need thereof, the method comprisingadministering to the subject an inhibitor of a protein tyrosine kinase(PTK) involved in cytokine signaling. In one embodiment, the inhibitoris a Jak1, Jak2, and/or a Jak3 inhibitor. In one embodiment, theinhibitor is a Stat1 and/or a Stat2 inhibitor. In a further embodiment,the inhibitor is INCB 018424. In one embodiment, the inhibitor istofacitinib (CP690550). In another embodiment, the hair-loss disordercomprises androgenetic alopecia, telogen effluvium, alopecia areata,telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis,hereditary hypotrichosis simplex, or alopecia universalis. In oneembodiment, the method further comprises determining whether theinhibitor administered induced hair growth in the subject afflicted witha hair loss disorder as compared to the subject's hair growth prior totreatment with the inhibitor. In one embodiment, the inhibitor is anantisense RNA that specifically inhibits expression of the gene thatencodes the Jak1 or Jak2 protein; a siRNA that specifically targets thethe gene that encodes the Jak1 or Jak2 protein; or a small molecule. Inone embodiment, the inhibitor is an antibody that specifically binds toa Jak3 protein or a fragment thereof; an antisense RNA or antisense DNAthat decreases expression of the gene that encodes the Jak3 protein; anantisense RNA or antisense DNA that decreases expression of the Jak3protein; a siRNA that specifically targets the Jak3 gene; a smallmolecule; or a combination thereof. In one embodiment, the smallmolecule is Janex 1 (WHI-P131), PF-956980, WHI-P154, tofacitinib(CP690550), VX-509, JAK3 Inhibitor IV, NSC114792, or R348. In someembodiments, the antibody specifically binds to a protein comprising SEQID NO: 109. In another embodiment, the siRNA is directed to a humannucleic acid sequence comprising SEQ ID NO: 110. In some embodiments,the siRNA directed to a Jak3 gene is any one of the sequences listed inTable 3. In another embodiment, the inhibitor is an antisense RNA thatspecifically inhibits expression of the gene that encodes the Stat1 orStat2 protein; a siRNA that specifically targets the the gene thatencodes the Stat1 or Stat2 protein; or a small molecule. In oneembodiment, the small molecule is AG490, CYT387, SB1518, LY3009104,TG101348, BMS-911543, or CEP-701. In another embodiment, the smallmolecule is WP-1034 (Faderl et al., Anticancer Res. 2005 May-June;25(3B):1841-50), fludarabine (Fludara, Berlex, Calif.),epigallocatechin-3-gallate (EGCG), or Hyperforin. In another embodiment,the siRNA is directed to a human nucleic acid sequence comprising SEQ IDNOS: 2, 4, 6 or 8. In some embodiments, the siRNA directed to a Jak1gene is any one of the sequences listed in Table 1. In otherembodiments, the siRNA directed to a Stat1 gene is any one of thesequences listed in Table 2. In a further embodiment, the administeringcomprises a subcutaneous, intra-muscular, intra-peritoneal, orintravenous injection; an infusion; oral, nasal, or topical delivery; ora combination thereof. In some embodiments, the administering occursdaily, weekly, twice weekly, monthly, twice monthly, or yearly. In someembodiments, the inhibitor, e.g., a Jak1, Jak2, and/or Jak3 inhibitor,is administered 1 time per week, 2 times per week, 3 times per week, 4times per week, 5 times per week, 6 times per week, 7 times per week, 8times per week, 9 times per week, 10 times per week, 9 times per week,10 times per week, 11 times per week, 12 times per week, 13 times perweek, or 14 times per week. In other embodiments, the subject isadministered the inhibitor for at least 1 week, at least 2 weeks, atleast 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, atleast 8 weeks, at least 12 weeks, or at least 16 weeks. In someembodiments, the method comprises administering a Jak1/2 inhibitor and aJak2 inhibitor to the subject. In further embodiments, administering theJak1/2 inhibitor is conducted simultaneously with the administering ofthe Jak3 inhibitor. Yet in other embodiments, administering the Jak1/2inhibitor is conducted sequentially in any order with the administeringof the Jak3 inhibitor. In some embodiments, the Jak1/2 inhibitor is INCB018424, GLPG0634, AG490, CYT387, SB 1518, LY3009104 (Baricitinib;INCB28050), AZD1480, TG101348, BMS-911543, or CEP-701.

An aspect of the invention provides for a method for inducing hairgrowth in a subject where the method comprises administering to thesubject an effective amount of an inhibitor of a protein tyrosine kinase(PTK) involved in cytokine signaling. In one embodiment, the inhibitoris a Jak1, Jak2, Jak3, and/or Stat inhibitor. In other embodiments, theinhibitor is INCB 018424. In one embodiment, the inhibitor is a Jak3inhibitor. In one embodiment, the inhibitor is tofacitinib (CP690550).In some embodiments, the subject is afflicted with a hair-loss disorder.In other embodiments, the hair-loss disorder comprises androgeneticalopecia, telogen effluvium, alopecia areata, telogen effluvium, tineacapitis, alopecia totalis, hypotrichosis, hereditary hypotrichosissimplex, or alopecia universalis. In some embodiments, the modulatingcompound can also inhibit hair growth, thus it can be used for treatmentof hair growth disorders, such as hypertrichosis. In one embodiment, themethod further comprises the step (b) determining whether the inhibitoradministered induced hair growth in the subject afflicted with a hairloss disorder as compared to the subject's hair growth prior totreatment with the inhibitor. In one embodiment, the inhibitor is anantisense RNA that specifically inhibits expression of the gene thatencodes the Jak1 or Jak2 protein; a siRNA that specifically targets thethe gene that encodes the Jak1 or Jak2 protein; or a small molecule. Inone embodiment, the inhibitor is an antibody that specifically binds toa Jak3 protein or a fragment thereof, an antisense RNA or antisense DNAthat decreases expression of the gene that encodes the Jak3 protein; anantisense RNA or antisense DNA that decreases expression of the Jak3protein; a siRNA that specifically targets the Jak3 gene; a smallmolecule; or a combination thereof. In one embodiment, the smallmolecule is Janex 1 (WHI-P131), PF-956980, WHI-P154, tofacitinib(CP690550), VX-509, JAK3 Inhibitor IV, NSC114792, or R348. In someembodiments, the antibody specifically binds to a protein comprising SEQID NO: 109. In another embodiment, the siRNA is directed to a humannucleic acid sequence comprising SEQ ID NO: 110. In some embodiments,the siRNA directed to a Jak3 gene is any one of the sequences listed inTable 3. In another embodiment, the inhibitor is an antisense RNA thatspecifically inhibits expression of the gene that encodes the Stat1 orStat2 protein; a siRNA that specifically targets the the gene thatencodes the Stat1 or Stat2 protein; or a small molecule. In oneembodiment, the small molecule is AG490, CYT387, SB1518, LY3009104,TG101348, BMS-911543, or CEP-701. In another embodiment, the smallmolecule is WP-1034 (Faderl et al., Anticancer Res. 2005 May-June;25(3B):1841-50), fludarabine (Fludara, Berlex, Calif.),epigallocatechin-3-gallate (EGCG), or Hyperforin. In another embodiment,the siRNA is directed to a human nucleic acid sequence comprising SEQ IDNOS: 2, 4, 6 or 8. In some embodiments, the siRNA directed to a Jak1gene is any one of the sequences listed in Table 1. In otherembodiments, the siRNA directed to a Stat1 gene is any one of thesequences listed in Table 2. In a further embodiment, the administeringcomprises a subcutaneous, intra-muscular, intra-peritoneal, orintravenous injection; an infusion; oral, nasal, or topical delivery; ora combination thereof. In some embodiments, the administering occursdaily, weekly, twice weekly, monthly, twice monthly, or yearly. In someembodiments, the inhibitor, e.g., a Jak1, Jak2, and/or Jak3 inhibitor,is administered 1 time per week, 2 times per week, 3 times per week, 4times per week, 5 times per week, 6 times per week, 7 times per week, 8times per week, 9 times per week, 10 times per week, 9 times per week,10 times per week, 11 times per week, 12 times per week, 13 times perweek, or 14 times per week. In other embodiments, the subject isadministered the inhibitor for at least 1 week, at least 2 weeks, atleast 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, atleast 8 weeks, at least 12 weeks, or at least 16 weeks. In someembodiments, the method comprises administering a Jak1/2 inhibitor and aJak2 inhibitor to the subject. In further embodiments, administering theJak1/2 inhibitor is conducted simultaneously with the administering ofthe Jak3 inhibitor. Yet in other embodiments, administering the Jak1/2inhibitor is conducted sequentially in any order with the administeringof the Jak3 inhibitor. In some embodiments, the Jak1/2 inhibitor is INCB018424, GLPG0634, AG490, CYT387, SB 1518, LY3009104 (Baricitinib;INCB28050), AZD1480, TG101348, BMS-911543, or CEP-701.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D are photographic images of clinical manifestations of AA. Inthe upper panels (FIGS. 1A-1B), patients with AA multiplex. In FIG. 1B,the patient is in regrowth phase. For patients with alopeciauniversalis, there is a complete lack of body hair and scalp hair (FIG.1C), while patients with alopecia totalis only lack scalp hair (FIG.1D). In FIG. 1D, hair regrowth is observed in the parietal region, whileno regrowth in either occipital or temporal regions is evident.

FIG. 2 shows a diagram showing the inflammatory response underlyingAlopecia Areata (AA).

FIG. 3 shows a diagram showing the inflammatory response underlyingAlopecia Areata (AA).

FIG. 4A is a photograph of lymph nodes and spleen isolated from C3H miceafflicted with AA (top) and control mice (bottom).

FIG. 4B show the results from FACS analysis that NKG2D bearing CD8 andTh1 cells are expanded in murine AA cutaneous lymph nodes.

FIG. 4C is a bar graph that shows a 6-fold increase in the number ofcytokine producing cells in C3H mice afflicted with AA as compared tocontrol mice.

FIG. 5A is a RNA signature of alopecic skin in C3H mice. The heat mapshows that 16 of the top 20 unregulated genes are Interferon-ResponseGenes. 16 of top 20 upregulated genes are Interferon Responsive Genes(IFN-γ but not Type I IFNs are elevated)

FIG. 5B shows transcriptional profiling of AA skin. qPCR verification ofsingle genes picked from the list generated shows an average fold changeover 3 experiments.

FIG. 6 is a diagram showing the targets in AA.

FIG. 7 shows a diagram showing the interferon γ pathway.

FIGS. 8A-8B show two gene mapping approaches. FIG. 8A shows linkageanalysis in families. FIG. 8B shows association analysis in a populationcohort.

FIG. 9 shows a Genomewide Association Study (GWAS) in AA.

FIGS. 10A-10B show danger signals: ULBP3 expression in the hair folliclein both control individuals (FIG. 10A) and AA patients (FIG. 10B).

FIGS. 11A-11B show that AA is caused by a “swarm of bees”: Killer CD8 Tcells attracted by NKG2DL. FIG. 11A shows “pre-2010” swarm of bees inAA. FIG. 11B shows “2012” identification of bees ((m) staining of CD8;(n) staining of NKG2D; and (o) colocalization of NKG2D and CD8).

FIG. 12 shows a diagram that shows the inflammatory response underlyingAA.

FIG. 13 shows INCB18424-202: Th1 and Th17 transcript markers and keratin16 are decreased with topical INCB18424, a JAK1 and JAK2 inhibitor,treatment.

FIG. 14 shows the Key Total Lesion Score results for INCB18424-203. Theresults how that Total Lesion Score for all dose groups ≥2× decreaseover vehicle, achieving significance after control for multiplicity

FIGS. 15A-15B show that treatment with INCB018424 markedly reducesepidermal thickening and dermal inflammation. No pathological skinthinning is observed.

FIG. 16 is a schematic that shows the pre-clinical strategies tointerrupt AA pathogenesis are aimed at interruption of the induction andeffector function of CD8 T cells and include blocking IFN-γ signals.

FIG. 17 shows that CD8+NKG2D+ T cells infiltrate AA hair follicles inboth mouse and human. Top panel: Human Alopecia Areata (Pethukova etal., 2010, Nature, 466(7302): 113-7); bottom panel-C3H/HeJ AlopeciaAreata.

FIG. 18-1, FIG. 18-2, and FIG. 18-3 show CD8+NKG2D+ T cells in alopecicC3H mice CD8□αβ⁺NKG2D⁺ T cells are massively expanded in the skin, lymphnode and blood of alopecic C3H mice. (NKG2D⁺ γδ T cells are present inboth affected and unaffected skin).

FIG. 19. shows CD8+NKG2D+ T cells in alopecic C3H mice. Immunophenotypeof gated LN CD8α⁺NKG2D⁺ populations shows that they are uniformlyCD8αβ⁺CD44⁺CXCR3⁺DX5³⁰ NKG2A/C/E+ IFN-γ producing memory T cells.

FIG. 20 shows spectratype analysis of C3H T cells in skin and LN:Similar TCR repertoires are shared by total skin and sorted CD8+NKG2D+LN T cells identifying these as the relevant effectors.

FIGS. 21A-21B show that IFN-γ producing CD8 T cells dominate human andmouse AA. FIG. 21A: Human: T cells obtained from T cell crawl-outs fromAA skin biopsies are uniformly CD8+NKG2D+ and produce IFN-γ uponPMA/Ionomycin stimulation. FIG. 21B: Mouse: C3H/HeJ stimulated lymphnode cells from alopecic mice contain 3 times more IFN-γ producing CD8and CD4 T cells than unaffected mice.

FIG. 22 shows that Type I and II IFNs are upregulated in C3H mouse AAskin: fold changes in expression of interferon genes (normalized to 18S)between 3 affected and 3 non affected C3H/HeJ mice, utilizing aninterferon signaling and response qPCR array (Stellarray Lonza Cat#00189608).

FIG. 23 shows graphs that show elevated serum chemokines and cytokinesin Human AA. Interferon-γ and IFN-induced chemokines (e.g.,IP-10/CXCL10) are elevated in the serum of human AA, in some casescorrelating with disease severity, i.e., patchy disease (AAP) vs.universalis (AU).

FIG. 24 is a luminex analysis that shows elevated IL-13 in the serum ofHuman AA subjects, in some cases correlating with disease severity,i.e., patchy disease (AAP) vs. universalis (AU).

FIG. 25 shows flow cytometry data that were gathered on a LSRII usingthe following mAbs in one tube to stain the entire population of C3Hdermal cells liberated from a 0.5 cm by 2 cm patch of alopecic skinafter 30 minutes collagenase digestion: (MHC II, CD11b, CD11c, CD19,120G8, CD3, CD4, CD8, DX5, NKG2D). γδNKG2D positive cells andCD8+DX5+NKG2D+ population are identified. These cells were also CD3+,with γδ□CD3 expression 100× higher than that seen in CD8+NK+ Tcells. >95% of the total CD3 positive cells were accounted.

FIG. 26 is a graph that shows the evaluation of PBMCs from four AAsubjects. NKG2D expression was increased in subsets of lymphocytes.

FIG. 27 shows spectratyping in a C3H-HeJ mouse. RNA was Isolated fromskin biopsies, and from sorted NKG2D- and NKG2D+CD8+ T cells fromcutaneous lymph nodes of C3H-HeJ mice. Spectratyping analysis shows CDR3length skewing in several Vbeta families. Some of the skewed Vbetafamilies are shared between skin and NKG2D+ lymph node CD8+ T cellpopulations (e.g., Vbeta2, Vbeta5.2, Vbeta20) whereas additional Vbetafamilies are skewed in skin (reflecting the CD4+ T cell population).

FIG. 28 is a schematic showing that human scalp skin procured fromcontrol or AA affected individuals can be used to establish primarycultures of individual cellular populations within the skin and hairfollicle.

FIGS. 29A-29D show a T cell ‘crawl-out’ assay, in which cultured humandermis obtained from punch skin biopsies are used as source material forflow cytometric analysis. Trisected skin punch biopsies of a 6 mm skinbiopsy is obtained from alopecic lesional and non-lesional areas. Afterremoving the fat, the finely minced skin is gently pressed in a Cellfoammatrix and then transferred, skin side-up, to a well of a 24-well platecontaining 2 mls of Skin T medium with IL-2 (6 ng/ml) and IL-15 (10ng/ml). T cells crawl outs are shown in FIG. 29A after the matrix isremoved 3 days later. Flow cytometry (FIGS. 29B-29C) and spectratyping(FIG. 29D) of these cells are also shown.

FIG. 30 shows that human peripheral blood mononuclear cells (PBMC), toppanels or murine splenocytes were cultured in high dose IL-2 (1000/ml)to generate lymphokine-activated killer (LAK) cells. The CFSE greenfluorescence labeled LAK cells were incubated with human hair follicleorgan culture (top panels), or mouse hair follicle culture (bottompanels) from non-lesion region (bottom left two) or lesion.

FIG. 31 shows immunohistochemical (IHC) staining of unaffected andaffected AA skin in C3H-HeJ mice.

FIG. 32 shows that treatment of mice with the Jak3 inhibitor(tofacitinib; CP-69055) prevents alopecia areata (AA).

FIGS. 33A, 33B-1, AND 33B-2 show treatment related elimination of dermalT cell infiltrates and inflammatory biomarkers by immunostaining (FIG.33A) and by flow cytometry (FIG. 33B-1-FIG. 33B-2). CD8+NKG2D+ T cells,which represent up to 25% of dermal cells in AA skin, are completelyeliminated with treatment. The flow cytometry is of single cellsuspensions from the skin. The cells in the right upper quadrant are notobserved in normal healthy mice and are CD8+NKG2D+ effector T cells.CD8+NKG2D+ effector T cells are found in alopecic mice after skingrafting. Mice treated with the JAK3 inhibitor do not develop alopeciaand their skin is devoid of these cells.

FIG. 34 are graphs that show treatment elimination of IFN-γ producing“NK-type” CD8 T cells in cutaneous LN, and loss of circulatingIFN-inducible chemokines. The bar graphs in the left panel showintracellular flow cytometry of PMA/Iono stimulated LN cells fromindividually treated and untreated mice stained forCD8.NKG2A/C/D/E.IFNγ. Two mice per group are shown.

FIG. 35 is a ribbon diagram that shows C3H/HeJ graft recipients weretreated with a JAK3 inhibitor (right panel), or PBS. RNA was extractedfrom skin harvested at weeks 6 (w6) and 13 (w13) following treatment andalso from mice that had undergone sham surgery and subsequentlyinterrogated by microarray analysis.

FIG. 36 shows photographs of mice before (top panel) and after topicaltreatment (9 days (middle panel) and 30 days (bottom panel)) with creamonly.

FIG. 37 shows photographs of mice before (top panel) and after topicaltreatment (9 days (middle panel) and 30 days (bottom panel)) with theJak3 inhibitor, tofacitinib.

FIG. 38 shows photographs of mice before (top panel) and after a controlinfusion (5 weeks (middle panel) and 10 weeks (bottom panel)) withoutthe Jak3 inhibitor, tofacitinib.

FIG. 39 shows photographs of mice before (top panel) and after a pumpinfusion (5 weeks (middle panel) and 10 weeks (bottom panel)) with theJak3 inhibitor, tofacitinib.

FIG. 40 shows treatment related elimination of dermal T cell infiltratesand inflammatory biomarkers by flow cytometry. The flow cytometry is oflymph node cells. The cells in the right upper quadrant are not observedin normal healthy mice and represent CD8+NKG2D+ effector T cells.CD8+NKG2D+ effector T cells are found in alopecic mice after skingrafting. Alopecic mice treated with the JAK3 inhibitor exhibit hairregrowth and their lymph nodes are devoid of these cells.

FIG. 41 shows that NKG2D “stress ligands” are upregulated in the hairfollicle, and demonstrates the shared pathogenesis in human and mouse.

FIG. 42 is a photomicrograph of an immunostain showing that CD8⁺ NKG2D⁺T cells infiltrate the hair follicle, and demonstrates the sharedpathogenesis in human and mouse.

FIG. 43 shows flow cytometric analysis that shows that NKG2D⁺CD8⁺ Tcells are the dominant infiltrating cells, and demonstrates the sharedpathogenesis in human and mouse. (e.g., see Clark, et al., (2006) JInvest Dermatol. 126(5): 1059-70, reporting a method for isolating Tcell crawlouts from the skin).

FIG. 44 shows that comparative transcriptomics reveals a shared IFNresponse signature, and demonstrates shared pathogenesis in human andmouse.

FIG. 45 shows plots depicting that CD8+NKG2D+ Cytotoxic T Cells areIFN-gamma producing cells bearing NK-type receptors.

FIG. 46 shows that NK Type CD8 T cells are required for T Cell transferof Alopecia Areata.

FIG. 47 shows pre-clinical studies using systemic JAK inhibitors in theC3H-HeJ mouse model of alopecia areata (for example, see O'Shea,Immunity. 2012 Apr. 20; 36(4):542-50).

FIG. 48 shows the results of pre-clinical studies with systemic deliveryof JAK3 inhibitor (tofacitinib), and tofacitinib treatment preventsAlopecia Areata.

FIG. 49 is a diagram depicting different examples of targeted therapiesfor alopecia areata. These topical therapies can be used to reverse theestablished disease.

FIG. 50 shows pre-clinical studies with topical delivery of JAK3inhibitor (tofacitinib). Topical treatment using JAK3 inhibitor resultsin reversal of long-standing AA (2-3 months duration). Before treatment,all mice had hair loss for 2-3 months. Control mice were applied withcream daily. Mice treated with the JAK3 inhibitor, Tofacitnib, weretreated daily with a topical cream containing 0.5% Tofacitnib.

FIG. 51 shows a summary of pre-clinical studies in alopecia C3H/HeJmice.

FIG. 52 shows the prevention of AA with systemic treatment with a JAK3inhibitor given at the time of skin grafting. All reagents are startedat time of graft (prevention). JAK3 inhibitor (CP690550) was deliveredusing Alzet osmotic mini-pumps (28-day pumps, Model 2004) implantedsubcutaneously, 10 mg/kg/day or vehicle (PEG300).

FIG. 53 shows photomicrographs depicting that treatment with Jak3inhibitor tofacitinib normalizes inflammatory infiltrate.

FIG. 54A shows the upregulation of NKG2DL in the hair follicle ofC3H/HeJ alopecic mice. Immunofluorescence staining of NKG2D ligands(H60) in the hair follicle inner root sheath (marked by K71).

FIG. 54B shows the cellular phenotype and function of CD8⁺NKG2D⁺cytotoxic T lymphocytes in AA mice. Immunofluorescence staining ofCD8⁺NKG2D⁺ cells in hair follicles of C57BL/6, healthy C3H/HeJ andC3H/HeJ AA mice.

FIG. 54C shows the cellular phenotype and function of CD8⁺NKG2D⁺cytotoxic T lymphocytes in AA mice. Striking cutaneous lymphadenopathyand cellularity in C3H/HeJ mice that developed AA.

FIG. 54D shows the cellular phenotype and function of CD8⁺NKG2D⁺cytotoxic T lymphocytes in AA mice. Plots show the frequency ofCD8⁺NKG2D⁺ T cells from skin and skin-draining lymph nodes in alopecicmice vs. ungrafted mice.

FIG. 54E shows the cellular phenotype and function of CD8⁺NKG2D⁺cytotoxic T lymphocytes in AA mice. Plots shows the immunophenotype ofCD8⁺NKG2D⁺ T cells in cutaneous lymph nodes in C3H/HeJ alopecic mice.

FIG. 54F shows the cellular phenotype and function of CD8⁺NKG2D⁺cytotoxic T lymphocytes in AA mice. Graphs demonstrate that CD8⁺NKG2D⁺ Tcells isolated from AA mice cutaneous LNs kill Rae-1 expressing dermalsheath cells grown from C3H/HeJ hair follicles in an NKG2D-dependentmanner.

FIG. 54G shows the cellular phenotype and function of CD8⁺NKG2D⁺cytotoxic T lymphocytes in AA mice. RNAseq was performed on CD8+NKG2D+and CD8+NKG2D− T cells sort purified from AA cutaneous lymph nodes.Transcripts upregulated in CD8+NKG2D+ T cells were compared with CTLgene expression and NK cell gene expression in the literature^(8,9), andoverlapping gene signatures are displayed in this Venn Diagram.

FIG. 54H shows the cellular phenotype and function of CD8⁺NKG2D⁺cytotoxic T lymphocytes in AA mice. C3H/HeJ mice were injectedsubcutaneously with 2×10⁶ cells from four different populations. Hairloss developed in recipients after either injection of total LN cells orCD8⁺NKG2D⁺ T cells alone (5 out of 5 mice per group), whereas micereceiving CD8⁺NKG2D⁻ T cells or LN cells depleted of NKG2D+ cells didnot develop alopecia (0 out of 5 mice per group). ***p value=<0.001.

FIG. 55A shows transcriptional profiling of human and mouse AA skin thatreveals evidence for IFNγ, cytotoxic T cells and the IL15 pathway ascentral to disease pathogenesis, inviting therapeutic targeting usingJak inhibitors. FIG. 55A is a Venn diagram showing overlap of gene listfrom whole alopecic C3H/HeJ mouse skin (compared with unaffected C3H/HeJskin) with IFNγ-induced gene expression¹¹ and CD8 and NK cell genesignatures from ImmGen consortium publications^(8,9) (Top panel).Representative list of differentially expressed genes among human AA andC3H/HeJ AA reveals shared inflammatory pathways, in particular IFNγpathway genes, genes representative of CD8 effectors, and a strikingIL-15 pathway signature (Bottom panel).

FIGS. 55B-55D show effect of treatment with a Jak3 inhibitor (JAK3i).FIG. 55B, JAK3i (tofacitinib) inhibits IL-15-induced Stat3 activation inT cells. FIG. 55C, JAK3i inhibits IL-15-induced LAK cell cytotoxicfunction. FIG. 55D, JAK3i inhibits IL-15-induced LAK cell Granzyme Bexpression and IFNγ production.

FIG. 56A shows targeting CD8⁺NKG2D⁺ cytotoxic T lymphocytes with JAK3iprevents the onset of alopecia in grafted C3H/HeJ mice. C3H/HeJ graftedmice were treated systemically from the time of grafting. The onset ofalopecia is inhibited by JAK3i, tofacitinib treatment. The durability ofdisease prevention was demonstrated by treatment withdrawal for anadditional 12 weeks after treatment withdrawal.

FIG. 56B shows targeting CD8⁺NKG2D⁺ cytotoxic T lymphocytes with JAK3iprevents the onset of alopecia in grafted C3H/HeJ mice. C3H/HeJ graftedmice were treated systemically from the time of grafting. The onset ofalopecia is inhibited by JAK3i, tofacitinib treatment.

FIG. 56C shows targeting CD8⁺NKG2D⁺ cytotoxic T lymphocytes with JAK3prevents the onset of alopecia in grafted C3H/HeJ mice. The frequency ofCD8⁺NKG2D⁺ T cells in skin and cutaneous lymph nodes of JAK3i treatedmice were significantly decreased compared to control mice. ***pvalue=<0.001.

FIG. 56D shows targeting CD8⁺NKG2D⁺ cytotoxic T lymphocytes with JAK3iprevents the onset of alopecia in grafted C3H/HeJ mice.Immunohistochemical staining of skin biopsies showed that the expressionof CD4, CD8, MHC class I and II in skin are significantly decreased inJAK3i treated mice compared to control mice.

FIG. 56E shows targeting CD8⁺NKG2D⁺ cytotoxic T lymphocytes with JAK3iprevents the onset of alopecia in grafted C3H/HeJ mice. ALADIN score (asummary statistic of expression of IFN signature and CTL signaturegenes) from JAK3i treated mice showed normalization. For gene expressionstudies, mice grafted with autologous health skin were included assham-operated controls.

FIG. 56F shows targeting CD8⁺NKG2D⁺ cytotoxic T lymphocytes with JAK3iprevents the onset of alopecia in grafted C3H/HeJ mice. GEDI results ofJAK3i treated mice showed reduction in CTL and IFN signatures.

FIG. 57A shows reversal of established AA with topical small moleculeinhibitor of the downstream effector kinase JAK3. Three-to-five pergroup of mice with long-standing AA (at least 12 weeks after grafting)were treated topically on the dorsal back with 0.5% JAK3i, tofacitinibor (top panels) vehicle alone (AQUAPHOR™) by daily application for 12weeks. A full coat of hair emerged in the JAK3i treated mice by 7 weeksof treatment and further developed by 12 weeks. The durability of hairregrowth was measured for an additional 8 weeks after treatmentwithdrawal with no evidence of disease recurrence.

FIG. 57B shows reversal of established AA with topical small moleculeinhibitor of the downstream effector kinase JAK3. The graph depicts atime course of hair regrowth index shown as weeks after treatment.

FIG. 57C shows reversal of established AA with topical small moleculeinhibitor of the downstream effector kinase, JAK3. The plots depict thefrequency of CD8⁺NKG2D⁺ T cells in the skin of JAK3i-treated mice wassignificantly decreased compared to control mice. *p value=<0.05, **pvalue=<0.01, n.s.=not significant.

FIG. 57D shows reversal of established AA with topical small moleculeinhibitor of the downstream effector kinase, JAK3. Immunohistochemicalstaining of skin biopsies shows treatment-related loss of expression ofCD4, CD8, MHC class I and II markers.

FIG. 57E shows reversal of established AA with topical small moleculeinhibitor of the downstream effector kinase, JAK3. The ALADIN scoreshows treatment-related loss of CTL and IFN signatures.

FIG. 57F shows reversal of established AA with topical small moleculeinhibitor of the downstream effector kinase, JAK3. GEDI analysis showsterritories of gene expression with treatment-related reversal ofpathogenic signatures.

FIG. 58A shows that Rae-1 is upregulated in the AA HF. Immunostaining oflesional using a pan-Rae-1 antibody in C3H/HeJ alopecic mice, unaffectedC3H/HeJ mice and C57B1.6 mice.

FIG. 58B shows that H60 and Rae-1 are overexpressed in AA. NKG2DL RNAexpression in alopecic lesional skin from C3H/HeJ mice compared withnon-lesional skin from unaffected C3H/HeJ mice. RT-PCR data from cDNAfrom 3 mice are shown and represented as relative fold-induction.

FIG. 58C shows that CD4 T cells are infrequent in AA lesional skin: Flowcytometric evaluation of lesional alopecic skin. Quantitation of CD4 andCD8 T cells as a percentage of total gated CD3⁺ or CD45⁺ cells.

FIG. 58D shows that effector memory immunophenotype of CD8⁺NKG2D⁺ Tcells are similar in lesional skin and in the cutaneous draining lymphnode. Gated CD8alpha⁺NKG2D⁺ T cells are displayed for CD8beta, NKG2D,NKG2A/C/E, CD44, CD103 and CD62L expression.

FIGS. 59, 59A-59D and 59-2 show a network map of differentiallyexpressed upregulated genes in CD3+CD8+NKG2D+ LN cells vs. CD8+NKG2D−cells. String.db was used to create a biological interaction scorematrix with the differentially expressed genes. The network map wascreated using cytoscape; only biological interactions >0.75 were used.Nodes represent genes, and edges represent biological interactions asderived from string.db. Node size is proportional to fold change, andedge width is proportional to biological interaction.

FIGS. 60A-60B show validation of mouse RNA expression studies. Todetermine the expression signature of C3H/HeJ mouse skin affected withalopecia areata, lesional skin was isolated from three affected femalemice and three unaffected aged-matched controls. Total and small RNAswere isolated from whole skin and biotin-labeled cRNA was generatedthrough in vitro transcription, followed by hybridization to theAffymetrix Mouse 430 2.0 Genechip. Data analysis was done as outlined inthe Methods. FIG. 60A is a heatmap depicting the significantly anddifferentially expressed genes between C3H/HeJ affected and unaffectedskin. FIG. 60B shows validation by qRT-PCR of several selectedimmune-related genes from this list whose expression levels aresignificantly upregulated in AA lesional skin compared to unaffectedskin, where each bar represents the average fold change of threeindependent experiments. UA=unaffected; AA=affected.

FIG. 61A shows systemic treatment of AA mice with JAK3 inhibitor.C3H/HeJ mice with long-standing alopecia areata were treated withtofacitinib with Alzet osmotic mini-pumps (pumps, model 2004, DurectCorporation) implanted subcutaneously on the back of each mouse todeliver vehicle (poly(ethylene glycol) (PEG)300) or vehicle containingJAK3i tofacitinib (Abmole) at 15 mg/kg/day for 12 wks. Alopecia areatareversal was complete on both the back and belly.

FIG. 61B shows systemic treatment of AA mice with JAK3 inhibitor. Flowcytometric analysis of skin and cutaneous lymph node populations showselimination of the CD8⁺NKG2D⁺ T cell population in treated mice (n=3 pergroup).

FIG. 61C shows systemic treatment of AA mice with JAK3 inhibitor.Immunostaining of skin from mice treated with tofactinib or placebodemonstrates elimination of CD8 infiltration and MHC I and IIupregulation in tofacitinib treated mice.

FIG. 62 depicts JAK-STAT signaling in normal hair follicle development.To analyze the status of the JAK-STAT signaling pathway during a normalhair cycle, mRNA was collected from skin of three mice at postnatal days17, 23, 29, 33. Samples represent the telogen to anagen transition asevident in the photomicrographs of H&E stains from these time points.Day 17=early telogen; day 23=late telogen; day 29=early anagen; day33=full anagen. cDNA was made and probed on JAK-STAT qPCR arrays.JAK-STAT signaling is up in telogen and down in anagen (see FIG. 63).

FIG. 63 is a heat map showing a hierarchical cluster analysis. mRNAisolated from mouse skin at each time point (D17, 23, 29, 33) wereprobed on JAK-STAT qPCR array. Red=induced, Green=repressed. Shown areall genes of the array. Several clusters of genes are evident that areupregulated in both telogen time points (d17 and d23), or upregulated inboth anagen time points (d29 and d33).

FIG. 64A is a heat map showing a hierarchical cluster analysis. Thefigure shows averages of 3 biological replicates/time point. The Controlgroup: D17 (Telogen); Group 1: D23 (telogen); Group 2: D29 (anagen); andGroup 3: D33 (anagen). JAK1 and 3 are upregulated in telogen group (redin control group and Group 1). Expression of JAK-STAT signaling pathwaycomponents is up in telogen and down in anagen.

FIG. 64B is a heat map showing a hierarchical cluster analysis. Thefigure shows averages of 3 biological replicates/time point. The Controlgroup: D17 (Telogen); Group 1: D23 (telogen); Group 2: D29 (anagen); andGroup 3: D33 (anagen). Stat 1/2/3/5 are upregulated in telogen (red incontrol group and group 1). Expression of JAK-STAT signaling pathwaycomponents is up in telogen and down in anagen.

FIG. 65 is a heat map (top) showing a cluster analysis. The Controlgroup: D17 (Telogen); Group 1: D23 (telogen); Group 2: D29 (anagen); andGroup 3: D33 (anagen). Potential ligands for JAK STAT or targets forJAK3i: OSM, IL6st (GP130), IL-4, IL-2Rg, CSF1R, and others upregulatedin telogen samples.

FIGS. 66-1-66-2 show summaries of genes upregulated in telogen as wellas downregulated in telogen. The Control group: D17 (Telogen); Group 1:D23 (telogen); Group 2: D29 (anagen); and Group 3: D33 (anagen). CRP, awell-defined gene upregulated by JAK/STAT signaling, is strikinglyincreased in telogen.

FIG. 67 are photomicrographs showing JAK-STAT in hair follicledevelopment: Embryonic development. Stat 3 and Stat 5 were identified tobe differentially expressed between telogen and anagen. To examine thepattern of expression in embryonic development, the activated(phosphorylated) forms of these proteins were stained. As shown, P-Stat3 is expressed in epithelial layers during early stages of developmentand then can be faintly seen in the dermal papilla. P-Stat5 expressionappears later, at E16.5, in scattered dermal cells and becomespronounced in the dermal condensate by E18.5.

FIG. 68 are photomicrographs showing JAK-STAT in hair follicledevelopment: post-natal development. P-Stat3 is expressed in the basallayers of the epidermis and upper epithelial layers of the follicleduring neonatal skin development. Expression can also be detected in thedermal papilla, most obviously during telogen (D17). P-Stat5 is highlyexpressed in the dermal papilla in all stage of the hair cycle. Intelogen, expression of P-Stat5 maybe limited to a subset of DP cells,closest to the K15+ bulge (D17, bottom).

FIG. 69 are photomicrographs showing the effect of a JAK-STAT inhibitoron the hair cycle of normal mice: Induction of anagen. Inhibition of theJAK-STAT pathway during telogen (by application of a JAK inhibitor)results in early onset of anagen. 7-8 weeks old animals in telogen weretreated with controls or JAK STAT inhibitors. Negative (vehicle alone;Left Panel) and positive (SAG=sonic hedgehog agonist; Middle panel)controls show that DMSO treatment alone does not induce anagen, whiletreatment with sonic hedgehog agonist results in early (4-7 days posttreatment) induction of anagen, as expected. Right: Treatment with theJak 3 inhibitor Tofacitinib (10 mg/mL) results in marked induction ofanagen after 2 weeks or 3 weeks of treatment compared to vehicle treatedmice which remain in telogen.

FIG. 70 are photomicrographs showing effects of drug treatment onkeratinocyte proliferation in vivo. Tofacitinib does not appear to causehyperpoliferation of the epidermis. Top: Krt6, a marker of keratinocyteproliferation typically expressed in the inner root sheath of thefollicle. Bottom: the effects of drug treatment on proliferation of thefollicle. In tofacitinib treated skin, proliferation in the secondarygerm occurs later, indicating that Tofacitinib can induce anagen.

FIG. 71 are plots showing JAK-STAT in hair cycle: a drug treatment inmice. Quantification of number and thickness of hair follicles in skintreated with the Jak3 inhibitor, Tofacitinib, and a Jak1/2 inhibitorRuxolitinib, as compared to plucked skin and vehicle-treated (DMSO).Both the numbers of follicles as well as the thickness of follicles isgreater in drug treated skin than in vehicle treated mice.

FIG. 72 are photomicrographs showing the effect of Jak inhibitors onhuman hair follicle morphogenesis: drug treatments of human fetal scalp.Human embryonic scalp at 20 weeks was obtained and treated in vitro with30 mg/mL of the Jak3 inhibitor, Tofacitinib, or vehicle alone. Skin washarvested and sectioned and assessed for hair follicle morphology. Hairsin treated skin appear more advanced in the first anagen ofmorphogenesis compared to DMSO treated scalp.

FIG. 73 shows topical treatment with a JAK inhibitor. Seven-week-oldnormal C57BL/6 mice were shaved in telogen and treated with 1% Jak3inhibitor (Tofacitinib; Right panel), 1.5 mg/ml Isopropyl unoprostone(LATISSE™; Middle Right panel), 100 uM SAG (shh agonist; Middle Leftpanel), or DMSO as vehicle (Left Panel) by daily application for 3weeks. Effect is durable out to a little more than 1 month at the timethe image was taken. Right half of each mouse was administered topicaldrug, left half of each mouse was treated with DMSO alone.

FIG. 74 are photomicrographs of Ki67 staining (green) showing markedproliferation in the telogen hair follicle in Tofacitinib and SAGtreated skin, compared to vehicle, indicative of the start of Anagen.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides for methods of treating a hair loss disorder(e.g., Alopecia Areata (AA), a common autoimmune form of hair loss) withan inhibitor of the Jak/Stat pathway, including Jak1, Jak2, Jak3, Stat1,and Stat2. Clinical research in AA has lagged behind its more heavilyinvestigated “sister” autoimmune diseases in which this gene has beenimplicated (e.g., rheumatoid arthritis (RA), type 1 diabetes mellitus(T1D), multiple sclerosis (MS)). The invention provides for therapeuticspreviously untested in AA, that can inform one about the clinicalrelevance of Jak/Stat pathways in AA and related diseases.

Abbreviations and Definitions

The singular forms “a”, “an” and “the” include plural reference unlessthe context clearly dictates otherwise.

As used herein the term “about” is used herein to mean approximately,roughly, around, or in the region of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent up or down(higher or lower).

Overview of the Integument and Hair Cells

The integument (or skin) is the largest organ of the body and is ahighly complex organ covering the external surface of the body. Itmerges, at various body openings, with the mucous membranes of thealimentary and other canals. The integument performs a number ofessential functions such as maintaining a constant internal environmentvia regulating body temperature and water loss; excretion by the sweatglands; but predominantly acts as a protective barrier against theaction of physical, chemical and biologic agents on deeper tissues. Skinis elastic and except for a few areas such as the soles, palms, andears, it is loosely attached to the underlying tissue. It also varies inthickness from 0.5 mm (0.02 inches) on the eyelids (“thin skin”) to 4 mm(0.17 inches) or more on the palms and soles (“thick skin”) (Ross M H,Histology: A text and atlas, 3^(rd) edition, Williams and Wilkins, 1995:Chapter 14; Burkitt H G, et al, Wheater's Functional Histology, 3^(rd)Edition, Churchill Livingstone, 1996: Chapter 9).

The skin is composed of two layers: a) the epidermis and b) the dermis.The epidermis is the outer layer, which is comparatively thin (0.1 mm).It is several cells thick and is composed of 5 layers: the stratumgerminativum, stratum spinosum, stratum granulosum, stratum lucidum(which is limited to thick skin), and the stratum corneum. The outermostepidermal layer (the stratum corneum) consists of dead cells that areconstantly shed from the surface and replaced from below by a single,basal layer of cells, called the stratum germinativum. The epidermis iscomposed predominantly of keratinocytes, which make up over 95% of thecell population. Keratinocytes of the basal layer (stratum germinativum)are constantly dividing, and daughter cells subsequently move upwardsand outwards, where they undergo a period of differentiation, and areeventually sloughed off from the surface. The remaining cell populationof the epidermis includes dendritic cells such as Langerhans cells andmelanocytes. The epidermis is essentially cellular and non-vascular,containing little extracellular matrix except for the layer of collagenand other proteins beneath the basal layer of keratinocytes (Ross M H,Histology: A text and atlas, 3^(rd) edition, Williams and Wilkins, 1995:Chapter 14; Burkitt H G, et al, Wheater's Functional Histology, 3^(rd)Edition, Churchill Livingstone, 1996: Chapter 9).

The dermis is the inner layer of the skin and is composed of a networkof collagenous extracellular material, blood vessels, nerves, andelastic fibers. Within the dermis are hair follicles with theirassociated sebaceous glands (collectively known as the pilosebaceousunit) and sweat glands. The interface between the epidermis and thedermis is extremely irregular and uneven, except in thin skin. Beneaththe basal epidermal cells along the epidermal-dermal interface, thespecialized extracellular matrix is organized into a distinct structurecalled the basement membrane (Ross M H, Histology: A text and atlas,3^(rd) edition, Williams and Wilkins, 1995: Chapter 14; Burkitt H G, etal, Wheater's Functional Histology, 3^(rd) Edition, ChurchillLivingstone, 1996: Chapter 9).

The mammalian hair fiber is composed of keratinized cells and developsfrom the hair follicle. The hair follicle is a peg of tissue derivedfrom a downgrowth of the epidermis, which lies immediately underneaththe skin's surface. The distal part of the hair follicle is in directcontinuation with the external, cutaneous epidermis. Although a smallstructure, the hair follicle comprises a highly organized system ofrecognizably different layers arranged in concentric series. Active hairfollicles extend down through the dermis, the hypodermis (which is aloose layer of connective tissue), and into the fat or adipose layer(Ross M H, Histology: A text and atlas, 3^(rd) edition, Williams andWilkins, 1995: Chapter 14; Burkitt H G, et al, Wheater's FunctionalHistology, 3^(rd) Edition, Churchill Livingstone, 1996: Chapter 9).

At the base of an active hair follicle lies the hair bulb. The bulbconsists of a body of dermal cells, known as the dermal papilla,contained in an inverted cup of epidermal cells known as the epidermalmatrix. Irrespective of follicle type, the germinative epidermal cellsat the very base of this epidermal matrix produce the hair fiber,together with several supportive epidermal layers. The lowermost dermalsheath is contiguous with the papilla basal stalk, from where the sheathcurves externally around all of the hair matrix epidermal layers as athin covering of tissue. The lowermost portion of the dermal sheath thencontinues as a sleeve or tube for the length of the follicle (Ross M H,Histology: A text and atlas, 3^(rd) edition, Williams and Wilkins, 1995:Chapter 14; Burkitt H G, et al, Wheater's Functional Histology, 3^(rd)Edition, Churchill Livingstone, 1996: Chapter 9).

Developing skin appendages, such as hair and feather follicles, rely onthe interaction between the epidermis and the dermis, the two layers ofthe skin. In embryonic development, a sequential exchange of informationbetween these two layers supports a complex series of morphogeneticprocesses, which results in the formation of adult follicle structures.However, in contrast to general skin dermal and epidermal cells, certainhair follicle cell populations, following maturity, retain theirembryonic-type interactive, inductive, and biosynthetic behaviors. Theseproperties can be derived from the very dynamic nature of the cyclicalproductive follicle, wherein repeated tissue remodeling necessitates ahigh level of dermal-epidermal interactive communication, which is vitalfor embryonic development and would be desirable in other forms oftissue reconstruction.

The hair fiber is produced at the base of an active follicle at a veryrapid rate. For example, follicles produce hair fibers at a rate 0.4 mmper day in the human scalp and up to 1.5 mm per day in the rat vibrissaor whiskers, which means that cell proliferation in the follicleepidermis ranks amongst the fastest in adult tissues (Malkinson F D andJ T Kearn, Int J Dermatol 1978, 17:536-551). Hair grows in cycles. Theanagen phase is the growth phase, wherein up to 90% of the hairfollicles said to be in anagen; catagen is the involuting or regressingphase which accounts for about 1-2% of the hair follicles; and telogenis the resting or quiescent phase of the cycle, which accounts for about10-14% of the hair follicles. The cycle's length varies on differentparts of the body.

Hair follicle formation and cycling is controlled by a balance ofinhibitory and stimulatory signals. The signaling cues are potentiatedby growth factors that are members of the TGFβ-BMP family. A prominentantagonist of the members of the TGFβ-BMP family is follistatin.Follistatin is a secreted protein that inhibits the action of variousBMPs (such as BMP-2, -4, -7, and -11) and activins by binding to saidproteins, and purportedly plays a role in the development of the hairfollicle (Nakamura M, et al., FASEB J, 2003, 17(3):497-9; Patel K Intl JBiochem Cell Bio, 1998, 30:1087-93; Ueno N, et al., PNAS, 1987,84:8282-86; Nakamura T, et al., Nature, 1990, 247:836-8; Iemura S, etal., PNAS, 1998, 77:649-52; Fainsod A, et al., Mech Dev, 1997, 63:39-50;Gamer L W, et al., Dev Biol, 1999, 208:222-32).

The deeply embedded end bulb, where local dermal-epidermal interactionsdrive active fiber growth, is the signaling center of the hair folliclecomprising a cluster of mesencgymal cells, called the dermal papilla(DP). This same region is also central to the tissue remodeling anddevelopmental changes involved in the hair fiber's or appendage'sprecise alternation between growth and regression phases. The DP, a keyplayer in these activities, appears to orchestrate the complex programof differentiation that characterizes hair fiber formation from theprimitive germinative epidermal cell source (Oliver R F, J Soc CosmetChem, 1971, 22:741-755; Oliver R F and C A Jahoda, Biology of Wool andHair (eds Roger et al.), 1971, Cambridge University Press:51-67;Reynolds A J and C A Jahoda, Development, 1992, 115:587-593; Reynolds AJ, et al., J Invest Dermatol, 1993, 101:634-38).

The lowermost dermal sheath (DS) arises below the basal stalk of thepapilla, from where it curves outwards and upwards. This dermal sheaththen externally encases the layers of the epidermal hair matrix as athin layer of tissue and continues upward for the length of thefollicle. The epidermally-derived outer root sheath (ORS) also continuesfor the length of the follicle, which lies immediately internal to thedermal sheath in between the two layers, and forms a specializedbasement membrane termed the glassy membrane. The outer root sheathconstitutes little more than an epidermal monolayer in the lowerfollicle, but becomes increasingly thickened as it approaches thesurface. The inner root sheath (IRS) forms a mold for the developinghair shaft. It comprises three parts: the Henley layer, the Huxleylayer, and the cuticle, with the cuticle being the innermost portionthat touches the hair shaft. The IRS cuticle layer is a single cellthick and is located adjacent to the hair fiber. It closelyinterdigitates with the hair fiber cuticle layer. The Huxley layer cancomprise up to four cell layers. The IRS Henley layer is the single celllayer that runs adjacent to the ORS layer (Ross M H, Histology: A textand atlas, 3^(rd) edition, Williams and Wilkins, 1995: Chapter 14;Burkitt H G, et al, Wheater's Functional Histology, 3^(rd) Edition,Churchill Livingstone, 1996: Chapter 9).

Alopecia Areata

Alopecia areata (AA) is one of the most prevalent autoimmune diseases,affecting approximately 4.6 million people in the US alone, includingmales and females across all ethnic groups, with a lifetime risk of 1.7%(1) In AA, autoimmunity develops against the hair follicle, resulting innon-scarring hair loss that can begin as patches, which can coalesce andprogress to cover the entire scalp (alopecia totalis, AT) or eventuallythe entire body (alopecia universalis, AU) (FIG. 1). AA was firstdescribed by Cornelius Celsus in 30 A.D., using the term “ophiasis”,which means “snake”, due to the sinuous path of hair loss as it spreadslowly across the scalp. Hippocrates first used the Greek word‘alopekia’ (fox mange), the modern day term “alopecia areata” was firstused by Sauvages in his Nosologica Medica, published in 1760 in Lyons,France.

Curiously, AA preferentially affects pigmented hair follicles in theanagen (growth) phase of the hair cycle, and when the hair regrows inpatches of AA, it frequently grows back white or colorless. Thephenomenon of ‘sudden whitening of the hair’ is therefore ascribed to AAwith an acute onset, and has been documented throughout history ashaving affected several prominent individuals at times of profoundgrief, stress or fear (2). Examples include Shahjahan, who upon thedeath of his wife in 1631 experienced acute whitening of his hair, andin his grief built the Taj Mahal in her honor. Sir Thomas More, authorof Utopia, who on the eve of his execution in 1535 was said to havebecome ‘white in both beard and hair’. The sudden whitening of the hairis believed to result from an acute attack upon the pigmented hairfollicles, leaving behind the white hairs unscathed.

Several clinical aspects of AA remain unexplained but can hold importantclues toward understanding pathogenesis. AA attacks hairs only aroundthe base of the hair follicles, which are surrounded by dense clustersof lymphocytes, resulting in the pathognomic ‘swarm of bees’ appearanceon histology. Based on these observations, it is postulated that asignal(s) in the pigmented, anagen hair follicle is emitted whichinvokes an acute or chronic immune response against the lower end of thehair follicle, leading to hair cycle perturbation, acute hair shedding,hair shaft anomalies and hair breakage. Despite these dramaticperturbations in the hair follicle, there is no permanent organdestruction and the possibility of hair regrowth remains if immuneprivilege can be restored.

Throughout history, AA has been considered at times to be a neurologicaldisease brought on by stress or anxiety, or as a result of an infectiousagent, or even hormonal dysfunction. The concept of agenetically-determined autoimmune mechanism as the basis for AA emergedduring the 20^(th) century from multiple lines of evidence. AA hairfollicles exhibit an immune infiltrate with activated Th, Tc and NKcells (3, 4) and there is a shift from a suppressive (Th2) to anautoimmune (Th1) cytokine response. The humanized model of AA, whichinvolves transfer of AA patient scalp onto immune-deficient SCID miceillustrates the autoimmune nature of the disease, since transfer ofdonor T-cells causes hair loss only when co-cultured with hair follicleor human melanoma homogenate (5, 6). Regulatory T cells which serve tomaintain immune tolerance are observed in lower numbers in AA tissue(7), and transfer of these cells to C3H/HeJ mice leads to resistance toAA (8). Although AA has long been considered exclusively as a T-cellmediated disease, in recent years, an additional mechanism of diseasehas been postulated. The hair follicle is defined as one of a select fewimmune privileged sites in the body, characterized by the presence ofextracellular matrix barriers to impede immune cell trafficking, lack ofantigen presenting cells, and inhibition of NK cell activity via thelocal production of immunosuppressive factors and reduced levels of MHCclass I expression (9). Thus, the notion of a ‘collapse of immuneprivilege’ has also been invoked as part of the mechanism by which AAcan arise. Support for a genetic basis for AA comes from multiple linesof evidence, including the observed heritability in first degreerelatives (10, 11), twin studies (12), and most recently, from theresults of our family-based linkage studies (13).

Treatment of Hair Loss Disorders

This invention provides for the discovery that a known therapeutic, forexample an inhibitor of a protein tyrosine kinase (PTK) involved incytokine signaling, such as JAK/STAT proteins Jak 1, Jak2, Jak3 Stat 1or Stat 2 (e.g., INCB 018424, tofacitinib (CP690550), Janex 1(WHI-P131), PF-956980, WHI-P154, VX-509, JAK3 Inhibitor IV, NSC114792,or R348), can be used for the the treatment of hair loss disorders.Non-limiting examples of hair loss disorders include: androgeneticalopecia, Alopecia areata, telogen effluvium, alopecia areata, alopeciatotalis, and alopecia universalis.

An aspect of the invention encompasses a method of treating a hair-lossdisorder in a mammalian subject in need thereof, the method comprisingadministering to the subject an inhibitor of a protein tyrosine kinase(PTK) involved in cytokine signaling. In one embodiment, the inhibitoris a Jak/Stat inhibitor. In a further embodiment, the inhibitor is INCB018424. In some embodiments, the Jak3 inhibitor is an antibody thatspecifically binds to a Jak3 protein or a fragment thereof; an antisenseRNA or antisense DNA that decreases expression of the gene that encodesthe Jak3 protein; an antisense RNA or antisense DNA that decreasesexpression of the Jak3 protein; a siRNA that specifically targets theJak3 gene; a small molecule; or a combination thereof. In oneembodiment, the inhibitor is a Jak3 inhibitor. In a further embodiment,the inhibitor is tofacitinib (CP690550). In a further embodiment, thesmall molecule is Janex 1 (WHI-P131), PF-956980, WHI-P154, VX-509, JAK3Inhibitor IV, NSC114792, or R348. In another embodiment, the hair-lossdisorder comprises androgenetic alopecia, telogen effluvium, alopeciaareata, telogen effluvium, tinea capitis, alopecia totalis,hypotrichosis, hereditary hypotrichosis simplex, or alopecia universalis

An aspect of the invention provides for a method for inducing hairgrowth in a subject where the method comprises administering to thesubject an effective amount of an inhibitor of a protein tyrosine kinase(PTK) involved in cytokine signaling. In one embodiment, the inhibitoris a Jak/Stat inhibitor. In other embodiments, the inhibitor is INCB018424. In some embodiments, the Jak3 inhibitor is an antibody thatspecifically binds to a Jak3 protein or a fragment thereof; an antisenseRNA or antisense DNA that decreases expression of the gene that encodesthe Jak3 protein; an antisense RNA or antisense DNA that decreasesexpression of the Jak3 protein; a siRNA that specifically targets theJak3 gene; a small molecule; or a combination thereof. In oneembodiment, the inhibitor is a Jak3 inhibitor. In a further embodiment,the inhibitor is tofacitinib (CP690550). In a further embodiment, thesmall molecule is Janex 1 (WHI-P131), PF-956980, WHI-P154, VX-509, JAK3Inhibitor IV, NSC114792, or R348. In some embodiments, the subject isafflicted with a hair-loss disorder. In other embodiments, the hair-lossdisorder comprises androgenetic alopecia, telogen effluvium, alopeciaareata, telogen effluvium, tinea capitis, alopecia totalis,hypotrichosis, hereditary hypotrichosis simplex, or alopeciauniversalis. In some embodiments, the modulating compound can alsoinhibit hair growth, thus it can be used for treatment of hair growthdisorders, such as hypertrichosis.

An aspect of the invention encompasses a method of treating a hair-lossdisorder in a mammalian subject in need thereof, the method comprisingadministering to the subject a Jak1 or Jak3 inhibitor. In oneembodiment, the inhibitor is an antibody or antibody fragment that isdirected to SEQ ID NO: 1 (Jak1), SEQ ID NO: 3 (Jak2), or SEQ ID NO: 109(Jak3). In another embodiment, the hair-loss disorder comprisesandrogenetic alopecia, telogen effluvium, alopecia areata, telogeneffluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditaryhypotrichosis simplex, or alopecia universalis.

An aspect of the invention provides for a method for inducing hairgrowth in a subject where the method comprises administering to thesubject an effective amount of a Jak1, Jak2, or Jak3 inhibitor, therebycontrolling hair growth in the subject. In one embodiment, the inhibitorcomprises an antibody that specifically binds to a protein comprisingSEQ ID NO: 1 (Jak1), SEQ ID NO: 3 (Jak2), or SEQ ID NO: 109 (Jak3). Insome embodiments, the subject is afflicted with a hair-loss disorder. Inother embodiments, the hair-loss disorder comprises androgeneticalopecia, telogen effluvium, alopecia areata, telogen effluvium, tineacapitis, alopecia totalis, hypotrichosis, hereditary hypotrichosissimplex, or alopecia universalis. In some embodiments, the modulatingcompound can also inhibit hair growth, thus it can be used for treatmentof hair growth disorders, such as hypertrichosis.

An aspect of the invention encompasses a method of treating a hair-lossdisorder in a mammalian subject in need thereof, the method comprisingadministering to the subject a Stat 1 inhibitor. In one embodiment, theinhibitor is an antibody or antibody fragment that is directed to SEQ IDNO: 5. In another embodiment, the hair-loss disorder comprisesandrogenetic alopecia, telogen effluvium, alopecia areata, telogeneffluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditaryhypotrichosis simplex, or alopecia universalis

An aspect of the invention provides for a method for inducing hairgrowth in a subject where the method comprises administering to thesubject an effective amount of a Stat 1 inhibitor, thereby controllinghair growth in the subject. In one embodiment, the inhibitor comprisesan antibody that specifically binds to a protein comprising SEQ ID NO:5. In some embodiments, the subject is afflicted with a hair-lossdisorder. In other embodiments, the hair-loss disorder comprisesandrogenetic alopecia, telogen effluvium, alopecia areata, telogeneffluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditaryhypotrichosis simplex, or alopecia universalis. In some embodiments, themodulating compound can also inhibit hair growth, thus it can be usedfor treatment of hair growth disorders, such as hypertrichosis.

An aspect of the invention encompasses a method of treating a hair-lossdisorder in a mammalian subject in need thereof, the method comprisingadministering to the subject a Stat 2 inhibitor. In one embodiment, theinhibitor is an antibody or antibody fragment that is directed to SEQ IDNO: 7. In another embodiment, the hair-loss disorder comprisesandrogenetic alopecia, telogen effluvium, alopecia areata, telogeneffluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditaryhypotrichosis simplex, or alopecia universalis

An aspect of the invention provides for a method for inducing hairgrowth in a subject where the method comprises administering to thesubject an effective amount of a Stat 2 inhibitor, thereby controllinghair growth in the subject. In one embodiment, the inhibitor comprisesan antibody that specifically binds to a protein comprising SEQ ID NO:7. In some embodiments, the subject is afflicted with a hair-lossdisorder. In other embodiments, the hair-loss disorder comprisesandrogenetic alopecia, telogen effluvium, alopecia areata, telogeneffluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditaryhypotrichosis simplex, or alopecia universalis. In some embodiments, themodulating compound can also inhibit hair growth, thus it can be usedfor treatment of hair growth disorders, such as hypertrichosis.

This invention provides for the discovery that a number human genes havebeen identified as a cohort of genes involved in telogen-to-anagentransition of the hair cycle (e.g, Telogen-to-Anagen Hair Cycle (TAHC)gene). These genes were identified as being upregulated in the telogenphase of the hair cycle, and can be correlated with the presence of ahair loss disorder in a subject. These genes, now that they have beenidentified, can be used for a variety of useful methods; for example,they can be used to determine whether a subject has susceptibility to ahair-loss disorder, such as Alopecia Areata (AA). The genes identifiedas part of this telogen-to-anagen transition hair cycle cohort or group(i.e., “TAHC genes”) include CSF1R (Gene ID Accession No. 1436), FCER2(Gene ID Accession No. 2208), IFNGR1 (Gene ID Accession No. 3459), IL20(Gene ID Accession No. 50604), OAS1 (Gene ID Accession No. 4938), PTPRC(Gene ID Accession No. 5788), CEBPD (Gene ID Accession No. 1052), CRP(Gene ID Accession No. 1401), IL2RA (Gene ID Accession No. 3559), IL4(Gene ID Accession No. 3565), IL6ST (Gene ID Accession No. 3572), INSR(Gene ID Accession No. 3643), JAK3 (Gene ID Accession No. 3718), NR3C1(Gene ID Accession No. 2908), OSM (Gene ID Accession No. 5008), PTPN11(Gene ID Accession No. 5781), SOCS3 (Gene ID Accession No. 9021), STAT5A(Gene ID Accession No. 6776), STAT5B (Gene ID Accession No. 6777), CCND1(Gene ID Accession No. 595), F2 (Gene ID Accession No. 2147), LRG1 (GeneID Accession No. 116844), PRLR (Gene ID Accession No. 5618), MPL (GeneID Accession No. 4352), and JUNB (Gene ID Accession No. 3726).

In one embodiment, the invention encompasses a method for detecting thepresence of or a predisposition to a hair-loss disorder in a humansubject where the method comprises obtaining a biological sample from ahuman subject; and detecting whether or not there is an alteration inthe level of expression of an mRNA or a protein encoded by a TAHC genein the subject as compared to the level of expression in a subject notafflicted with a hair-loss disorder. In on embodiment, the detectingcomprises determining whether mRNA expression or protein expression ofthe TAHC gene is increased or decreased as compared to expression in anormal sample. In another embodiment, the detecting comprisesdetermining in the sample whether expression of at least 2 TAHCproteins, at least 3 TAHC proteins, at least 4 TAHC proteins, at least 5TAHC proteins, at least 6 TAHC proteins, at least 6 TAHC proteins, atleast 7 TAHC proteins, or at least 8 TAHC proteins is increased ordecreased as compared to expression in a normal sample. In someembodiments, the detecting comprises determining in the sample whetherexpression of at least 2 TAHC mRNAs, at least 3 TAHC mRNAs, at least 4TAHC mRNAs, at least 5 TAHC mRNAs, at least 6 TAHC mRNAs, at least 6TAHC mRNAs, at least 7 TAHC mRNAs, or at least 8 TAHC mRNAs is increasedor decreased as compared to expression in a normal sample. In oneembodiment, an increase in the expression of at least 2 TAHC genes, atleast 3 TAHC genes, at least 4 TAHC genes, at least 5 TAHC genes, atleast 6 TAHC genes, at least 7 TAHC genes, or at least 8 TAHC genesindicates a predisposition to or presence of a hair-loss disorder in thesubject. In another embodiment, a decrease in the expression of at least2 TAHC genes, at least 3 TAHC genes, at least 4 TAHC genes, at least 5TAHC genes, at least 6 TAHC genes, at least 7 TAHC genes, or at least 8TAHC genes indicates a predisposition to or presence of a hair-lossdisorder in the subject. In one embodiment, the mRNA expression orprotein expression level in the subject is about 5-fold increased, about10-fold increased, about 15-fold increased, about 20-fold increased,about 25-fold increased, about 30-fold increased, about 35-foldincreased, about 40-fold increased, about 45-fold increased, about50-fold increased, about 55-fold increased, about 60-fold increased,about 65-fold increased, about 70-fold increased, about 75-foldincreased, about 80-fold increased, about 85-fold increased, about90-fold increased, about 95-fold increased, or is 100-fold increased, ascompared to that in the normal sample. In some embodiments, the he mRNAexpression or protein expression level in the subject is at least about100-fold increased, at least about 200-fold increased, at least about300-fold increased, at least about 400-fold increased, or is at leastabout 500-fold increased, as compared to that in the normal sample. Infurther embodiments, the mRNA expression or protein expression level ofthe TAHC gene in the subject is about 5-fold to about 70-fold increased,as compared to that in the normal sample. In other embodiments, the mRNAor protein expression level of the TAHC gene in the subject is about5-fold to about 90-fold increased, as compared to that in the normalsample. In one embodiment, the mRNA expression or protein expressionlevel in the subject is about 5-fold decreased, about 10-fold decreased,about 15-fold decreased, about 20-fold decreased, about 25-folddecreased, about 30-fold decreased, about 35-fold decreased, about40-fold decreased, about 45-fold decreased, about 50-fold decreased,about 55-fold decreased, about 60-fold decreased, about 65-folddecreased, about 70-fold decreased, about 75-fold decreased, about80-fold decreased, about 85-fold decreased, about 90-fold decreased,about 95-fold decreased, or is 100-fold decreased, as compared to thatin the normal sample. In some embodiments, the mRNA expression orprotein expression level in the subject is at least about 100-folddecreased, as compared to that in the normal sample. In someembodiments, the mRNA or protein expression level of the TAHC gene inthe subject is about 5-fold to about 70-fold decreased, as compared tothat in the normal sample. In yet other embodiments, the mRNA or proteinexpression level of the TAHC gene in the subject is about 5-fold toabout 90-fold decreased, as compared to that in the normal sample. Infurther embodiments, the detecting comprises gene sequencing, selectivehybridization, selective amplification, gene expression analysis, or acombination thereof. In another embodiment, the hair-loss disordercomprises androgenetic alopecia, alopecia areata, telogen effluvium,alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, oralopecia universalis. In one embodiment, the TAHC gene is CSF1R, FCER2,IFNGR1, IL20, OAS1, PTPRC, CEBPD, CRP, IL2RA, IL4, IL6ST, INSR, JAK3,NR3C1, OSM, PTPN11, SOCS3, STAT5A, STAT5B, CCND1, F2, LRG1, PRLR, MPL,or JUNB. In another embodiment, the TAHC gene is CRP.

Diagnosis

The invention provides methods to diagnose whether or not a subject issusceptible to or has a hair loss disorder. The diagnostic methods, inone embodiment, are based on monitoring the expression of TAHC genes,such as CSF1R, FCER2, IFNGR1, IL20, OAS 1, PTPRC, CEBPD, CRP, IL2RA,IL4, IL6ST, INSR, JAK3, NR3C1, OSM, PTPN11, SOCS3, STAT5A, STAT5B,CCND1, F2, LRG1, PRLR, MPL, or JUNB, in a subject, for example whetherthey are increased or decreased as compared to a normal sample. As usedherein, the term “diagnosis” includes the detection, typing, monitoring,dosing, comparison, at various stages, including early, pre-symptomaticstages, and late stages, in adults and children. Diagnosis can includethe assessment of a predisposition or risk of development, theprognosis, or the characterization of a subject to define mostappropriate treatment (pharmacogenetics).

The invention provides diagnostic methods to determine whether anindividual is at risk of developing a hair-loss disorder, or suffersfrom a hair-loss disorder, wherein the disease results from analteration in the expression of TAHC genes. In one embodiment, a methodof detecting the presence of or a predisposition to a hair-loss disorderin a subject is provided. The subject can be a human or a child thereof.The method can comprise detecting in a sample from the subject whetheror not there is an alteration in the level of expression of a proteinencoded by a TAHC gene in the subject as compared to the level ofexpression in a subject not afflicted with a hair-loss disorder. In oneembodiment, the detecting can comprise determining whether mRNAexpression of the TAHC is increased or decreased. For example, in amicroarray assay, one can look for differential expression of a TAHCgene. Any expression of a TAHC gene that is either 2× higher or 2× lowerthan TAHC expression expression observed for a subject not afflictedwith a hair-loss disorder (as indicated by a fluorescent read-out) isdeemed not normal, and worthy of further investigation. The detectingcan also comprise determining in the sample whether expression of atleast 2 TAHC proteins, at least 3 TAHC proteins, at least 4 TAHCproteins, at least 5 TAHC proteins, at least 6 TAHC proteins, at least 6TAHC proteins, at least 7 TAHC proteins, or at least 8 TAHC proteins isincreased or decreased. The presence of such an alteration is indicativeof the presence or predisposition to a hair-loss disorder.

The presence of an alteration in a TAHC gene in the sample is detectedthrough the genotyping of a sample, for example via gene sequencing,selective hybridization, amplification, gene expression analysis, or acombination thereof. In one embodiment, the sample can comprise blood,serum, sputum, lacrimal secretions, semen, vaginal secretions, fetaltissue, skin tissue, epithelial tissue, muscle tissue, amniotic fluid,or a combination thereof.

The invention provides for a diagnostic kit used to determine whether asample from a subject exhibits increased expression of at least 2 ormore TAHC genes. In one embodiment, the kit comprising a nucleic acidprimer that specifically hybridizes to one or more TAHC genes. Theinvention also provides for a diagnostic kit used to determine whether asample from a subject exhibits a predisposition to a hair-loss disorderin a human subject. In further embodiments, the TAHC gene is CSF1R,FCER2, IFNGR1, IL20, OAS1, PTPRC, CEBPD, CRP, IL2RA, IL4, IL6ST, INSR,JAK3, NR3C1, OSM, PTPN11, SOCS3, STAT5A, STAT5B, CCND1, F2, LRG1, PRLR,MPL, or JUNB. In another embodiment, the hair-loss disorder comprisesandrogenetic alopecia, alopecia areata, telogen effluvium, alopeciatotalis, hypotrichosis, hereditary hypotrichosis simplex, or alopeciauniversalis.

DNA and Amino Acid Manipulation Methods and Purification Thereof

The present invention utilizes conventional molecular biology,microbiology, and recombinant DNA techniques available to one ofordinary skill in the art. Such techniques are well known to the skilledworker and are explained fully in the literature. See, e.g., Maniatis,Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual” (1982):“DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover,ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984);“Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985);“Transcription and Translation” (B. D. Hames & S. J. Higgins, eds.,1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “ImmobilizedCells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide toMolecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: aLaboratory Manual” (1989).

One skilled in the art can obtain a protein in several ways, whichinclude, but are not limited to, isolating the protein via biochemicalmeans or expressing a nucleotide sequence encoding the protein ofinterest by genetic engineering methods.

A protein is encoded by a nucleic acid (including, for example, genomicDNA, complementary DNA (cDNA), synthetic DNA, as well as any form ofcorresponding RNA). For example, it can be encoded by a recombinantnucleic acid of a gene. The proteins of the invention can be obtainedfrom various sources and can be produced according to various techniquesknown in the art. For example, a nucleic acid that encodes a protein canbe obtained by screening DNA libraries, or by amplification from anatural source. A protein can be a fragment or portion thereof. Thenucleic acids encoding a protein can be produced via recombinant DNAtechnology and such recombinant nucleic acids can be prepared byconventional techniques, including chemical synthesis, geneticengineering, enzymatic techniques, or a combination thereof. Forexample, a Jak 1 protein is the polypeptide encoded by the nucleic acidhaving the nucleotide sequence shown in SEQ ID NO: 2. An example of aJak 1 polypeptide has the amino acid sequence shown in SEQ ID NO: 1. AJak 2 protein is the polypeptide encoded by the nucleic acid having thenucleotide sequence shown in SEQ ID NO: 4. An example of a Jak 2polypeptide has the amino acid sequence shown in SEQ ID NO: 3. A Jak 3protein is the polypeptide encoded by the nucleic acid having thenucleotide sequence shown in SEQ ID NO: 110. An example of a Jak 3polypeptide has the amino acid sequence shown in SEQ ID NO: 109. Forexample, a Stat 1 protein is the polypeptide encoded by the nucleic acidhaving the nucleotide sequence shown in SEQ ID NO: 6. An example of aStat 1 polypeptide has the amino acid sequence shown in SEQ ID NO: 5. AStat 2 protein is the polypeptide encoded by the nucleic acid having thenucleotide sequence shown in SEQ ID NO: 8. An example of a Stat 2polypeptide has the amino acid sequence shown in SEQ ID NO: 7.

The polypeptide sequence of human Jak 1 is depicted in SEQ ID NO: 1. Thenucleotide sequence of human Jak 1 is shown in SEQ ID NO: 2. Sequenceinformation related to Jak 1 is accessible in public databases byGenBank Accession numbers NP_002218 (for protein) and NM_002227.2 (fornucleic acid).

SEQ ID NO: 1 is the human wild type amino acid sequencecorresponding to Jak1 (residues 1-1154): 1MQYLNIKEDC NAMAFCAKMR SSKKTEVNLE APEPGVEVIF YLSDREPLRL GSGEYTAEEL 61CIRAAQACRI SPLCHNLFAL YDENTKLWYA PNRTITVDDK MSLRLHYRMR FYFTNWHGTN 121DNEQSVWRHS PKKQKNGYEK KKIPDATPLL DASSLEYLFA QGQYDLVKCL APIRDPKTEQ 181DGHDIENECL GMAVLAISHY AMMKKMQLPE LPKDISYKRY IPETLNKSIR QRNLLTRMRI 241NNVFKDFLKE FNNKTICDSS VSTHDLKVKY LATLETLTKH YGAEIFETSM LLISSENEMN 301WFHSNDGGNV LYYEVMVTGN LGIQWRHKPN VVSVEKEKNK LKRKKLENKH KKDEEKNKIR 361EEWNNFSYFP EITHIVIKES VVSINKQDNK KMELKLSSHE EALSFVSLVD GYFRLTADAH 421HYLCTDVAPP LIVHNIQNGC HGPICTEYAI NKLRQEGSEE GMYVLRWSCT DFDNILMTVT 481CFEKSEQVQG AQKQFKNFQI EVQKGRYSLH GSDRSFPSLG DLMSHLKKQI LRTDNISFML 541KRCCQPKPRE ISNLLVATKK AQEWQPVYPM SQLSFDRILK KDLVQGEHLG RGTRTHIYSG 601TLMDYKDDEG TSEEKKIKVI LKVLDPSHRD ISLAFFEAAS MMRQVSHKHI VYLYGVCVRD 661VENIMVEEFV EGGPLDLFMH RKSDVLTTPW KFKVAKQLAS ALSYLEDKDL VHGNVCTKNL 721LLAREGIDSE CGPFIKLSDP GIPITVLSRQ ECIERIPWIA PECVEDSKNL SVAADKWSFG 781TTLWEICYNG EIPLKDKTLI EKERFYESRC RPVTPSCKEL ADLMTRCMNY DPNQRPFFRA 841IMRDINKLEE QNPDIVSEKK PATEVDPTHF EKRFLKRIRD LGEGHFGKVE LCRYDPEGDN 901TGEQVAVKSL KPESGGNHIA DLKKEIEILR NLYHENIVKY KGICTEDGGN GIKLIMEFLP 961SGSLKEYLPK NKNKINLKQQ LKYAVQICKG MDYLGSRQYV HRDLAARNVL VESEHQVKIG 1021DFGLTKAIET DKEYYTVKDD RDSPVFWYAP ECLMQSKFYI ASDVWSFGVT LHELLTYCDS 1081DSSPMALFLK MIGPTHGQMT VTRLVNTLKE GKRLPCPPNC PDEVYQLMRK CWEFQPSNRT 1141SFQNLIEGFE ALLK SEQ ID NO: 2 is the human wild type nucleotide sequencecorresponding to Jak1 (nucleotides 1-5053), wherein the underscoredbolded “ATG” denotes the beginning of the open reading frame: 1tgcagacagt gcgggcctgc gcccagtccc ggctgtcctc gccgcgaccc ctcctcagcc 61ctgggcgcgc gcacgctggg gccccgcggg gctggccgcc tagcgagcct gccggtcgac 121cccagccagc gcagcgacgg ggcgctgcct ggcccaggcg cacacggaag tgcgcttctc 181tgaagtagct ttggaaagta gagaagaaaa tccagtttgc ttcttggaga acactggaca 241gctgaataa a tg cagtatct aaatataaaa gaggactgca atgccatggc tttctgtgct 301aaaatgagga gctccaagaa gactgaggtg aacctggagg cccctgagcc aggggtggaa 361gtgatcttct atctgtcgga cagggagccc ctccggctgg gcagtggaga gtacacagca 421gaggaactgt gcatcagggc tgcacaggca tgccgtatct ctcctctttg tcacaacctc 481tttgccctgt atgacgagaa caccaagctc tggtatgctc caaatcgcac catcaccgtt 541gatgacaaga tgtccctccg gctccactac cggatgaggt tctatttcac caattggcat 601ggaaccaacg acaatgagca gtcagtgtgg cgtcattctc caaagaagca gaaaaatggc 661tacgagaaaa aaaagattcc agatgcaacc cctctccttg atgccagctc actggagtat 721ctgtttgctc agggacagta tgatttggtg aaatgcctgg ctcctattcg agaccccaag 781accgagcagg atggacatga tattgagaac gagtgtctag ggatggctgt cctggccatc 841tcacactatg ccatgatgaa gaagatgcag ttgccagaac tgcccaagga catcagctac 901aagcgatata ttccagaaac attgaataag tccatcagac agaggaacct tctcaccagg 961atgcggataa ataatgtttt caaggatttc ctaaaggaat ttaacaacaa gaccatttgt 1021gacagcagcg tgtccacgca tgacctgaag gtgaaatact tggctacctt ggaaactttg 1081acaaaacatt acggtgctga aatatttgag acttccatgt tactgatttc atcagaaaat 1141gagatgaatt ggtttcattc gaatgacggt ggaaacgttc tctactacga agtgatggtg 1201actgggaatc ttggaatcca gtggaggcat aaaccaaatg ttgtttctgt tgaaaaggaa 1261aaaaataaac tgaagcggaa aaaactggaa aataaacaca agaaggatga ggagaaaaac 1321aagatccggg aagagtggaa caatttttct tacttccctg aaatcactca cattgtaata 1381aaggagtctg tggtcagcat taacaagcag gacaacaaga aaatggaact gaagctctct 1441tcccacgagg aggccttgtc ctttgtgtcc ctggtagatg gctacttccg gctcacagca 1501gatgcccatc attacctctg caccgacgtg gcccccccgt tgatcgtcca caacatacag 1561aatggctgtc atggtccaat ctgtacagaa tacgccatca ataaattgcg gcaagaagga 1621agcgaggagg ggatgtacgt gctgaggtgg agctgcaccg actttgacaa catcctcatg 1681accgtcacct gctttgagaa gtctgagcag gtgcagggtg cccagaagca gttcaagaac 1741tttcagatcg aggtgcagaa gggccgctac agtctgcacg gttcggaccg cagcttcccc 1801agcttgggag acctcatgag ccacctcaag aagcagatcc tgcgcacgga taacatcagc 1861ttcatgctaa aacgctgctg ccagcccaag ccccgagaaa tctccaacct gctggtggct 1921actaagaaag cccaggagtg gcagcccgtc taccccatga gccagctgag tttcgatcgg 1981atcctcaaga aggatctggt gcagggcgag caccttggga gaggcacgag aacacacatc 2041tattctggga ccctgatgga ttacaaggat gacgaaggaa cttctgaaga gaagaagata 2101aaagtgatcc tcaaagtctt agaccccagc cacagggata tttccctggc cttcttcgag 2161gcagccagca tgatgagaca ggtctcccac aaacacatcg tgtacctcta tggcgtctgt 2221gtccgcgacg tggagaatat catggtggaa gagtttgtgg aagggggtcc tctggatctc 2281ttcatgcacc ggaaaagcga tgtccttacc acaccatgga aattcaaagt tgccaaacag 2341ctggccagtg ccctgagcta cttggaggat aaagacctgg tccatggaaa tgtgtgtact 2401aaaaacctcc tcctggcccg tgagggcatc gacagtgagt gtggcccatt catcaagctc 2461agtgaccccg gcatccccat tacggtgctg tctaggcaag aatgcattga acgaatccca 2521tggattgctc ctgagtgtgt tgaggactcc aagaacctga gtgtggctgc tgacaagtgg 2581agctttggaa ccacgctctg ggaaatctgc tacaatggcg agatcccctt gaaagacaag 2641acgctgattg agaaagagag attctatgaa agccggtgca ggccagtgac accatcatgt 2701aaggagctgg ctgacctcat gacccgctgc atgaactatg accccaatca gaggcctttc 2761ttccgagcca tcatgagaga cattaataag cttgaagagc agaatccaga tattgtttca 2821gaaaaaaaac cagcaactga agtggacccc acacattttg aaaagcgctt cctaaagagg 2881atccgtgact tgggagaggg ccactttggg aaggttgagc tctgcaggta tgaccccgaa 2941ggggacaata caggggagca ggtggctgtt aaatctctga agcctgagag tggaggtaac 3001cacatagctg atctgaaaaa ggaaatcgag atcttaagga acctctatca tgagaacatt 3061gtgaagtaca aaggaatctg cacagaagac ggaggaaatg gtattaagct catcatggaa 3121tttctgcctt cgggaagcct taaggaatat cttccaaaga ataagaacaa aataaacctc 3181aaacagcagc taaaatatgc cgttcagatt tgtaagggga tggactattt gggttctcgg 3241caatacgttc accgggactt ggcagcaaga aatgtccttg ttgagagtga acaccaagtg 3301aaaattggag acttcggttt aaccaaagca attgaaaccg ataaggagta ttacaccgtc 3361aaggatgacc gggacagccc tgtgttttgg tatgctccag aatgtttaat gcaatctaaa 3421ttttatattg cctctgacgt ctggtctttt ggagtcactc tgcatgagct gctgacttac 3481tgtgattcag attctagtcc catggctttg ttcctgaaaa tgataggccc aacccatggc 3541cagatgacag tcacaagact tgtgaatacg ttaaaagaag gaaaacgcct gccgtgccca 3601cctaactgtc cagatgaggt ttatcaactt atgaggaaat gctgggaatt ccaaccatcc 3661aatcggacaa gctttcagaa ccttattgaa ggatttgaag cacttttaaa ataagaagca 3721tgaataacat ttaaattcca cagattatca agtccttctc ctgcaacaaa tgcccaagtc 3781attttttaaa aatttctaat gaaagaagtt tgtgttctgt ccaaaaagtc actgaactca 3841tacttcagta catatacatg tataaggcac actgtagtgc ttaatatgtg taaggacttc 3901ctctttaaat ttggtaccag taacttagtg acacataatg acaaccaaaa tatttgaaag 3961cacttaagca ctcctccttg tggaaagaat ataccaccat ttcatctggc tagttcacca 4021tcacaactgc attaccaaaa ggggattttt gaaaacgagg agttgaccaa aataatatct 4081gaagatgatt gcttttccct gctgccagct gatctgaaat gttttgctgg cacattaatc 4141atagataaag aaagattgat ggacttagcc ctcaaatttc agtatctata cagtactaga 4201ccatgcattc ttaaaatatt agataccagg tagtatatat tgtttctgta caaaaatgac 4261tgtattctct caccagtagg acttaaactt tgtttctcca gtggcttagc tcctgttcct 4321ttgggtgatc actagcaccc atttttgaga aagctggttc tacatggggg gatagctgtg 4381gaatagataa tttgctgcat gttaattctc aagaactaag cctgtgccag tgctttccta 4441agcagtatac ctttaatcag aactcattcc cagaacctgg atgctattac acatgctttt 4501aagaaacgtc aatgtatatc cttttataac tctaccactt tggggcaagc tattccagca 4561ctggttttga atgctgtatg caaccagtct gaataccaca tacgctgcac tgttcttaga 4621gggtttccat acttaccacc gatctacaag ggttgatccc tgtttttacc atcaatcatc 4681accctgtggt gcaacacttg aaagacccgg ctagaggcac tatggacttc aggatccact 4741agacagtttt cagtttgctt ggaggtagct gggtaatcaa aaatgtttag tcattgattc 4801aatgtgaacg attacggtct ttatgaccaa gagtctgaaa atctttttgt tatgctgttt 4861agtattcgtt tgatattgtt acttttcacc tgttgagccc aaattcagga ttggttcagt 4921ggcagcaatg aagttgccat ttaaatttgt tcatagccta catcaccaag gtctctgtgt 4981caaacctgtg gccactctat atgcactttg tttactcttt atacaaataa atatactaaa 5041gactttacat gca

The polypeptide sequence of human Jak 2 is depicted in SEQ ID NO: 3. Thenucleotide sequence of human Jak 2 is shown in SEQ ID NO: 4. Sequenceinformation related to Jak 2 is accessible in public databases byGenBank Accession numbers NP_004963 (for protein) and NM_004972.3 (fornucleic acid).

SEQ ID NO: 3 is the human wild type amino acid sequence corresponding toJak2 (residues 1-1132): 1 MGMACLTMTE MEGTSTSSIY QNGDISGNAN SMKQIDPVLQVYLYHSLGKS EADYLTFPSG 61 EYVAEEICIA ASKACGITPV YHNMFALMSE TERIWYPPNHVFHIDESTRH NVLYRIRFYF 121 PRWYCSGSNR AYRHGISRGA EAPLLDDFVM SYLFAQWRHDFVHGWIKVPV THETQEECLG 181 MAVLDMMRIA KENDQTPLAI YNSISYKTFL PKCIRAKIQDYHILTRKRIR YRFRRFIQQF 241 SQCKATARNL KLKYLINLET LQSAFYTEKF EVKEPGSGPSGEEIFATIII TGNGGIQWSR 301 GKHKESETLT EQDLQLYCDF PNIIDVSIKQ ANQEGSNESRVVTIHKQDGK NLEIELSSLR 361 EALSFVSLID GYYRLTADAH HYLCKEVAPP AVLENIQSNCHGPISMDFAI SKLKKAGNQT 421 GLYVLRCSPK DFNKYFLTFA VERENVIEYK HCLITKNENEEYNLSGTKKN FSSLKDLLNC 481 YQMETVRSDN IIFQFTKCCP PKPKDKSNLL VFRTNGVSDVPTSPTLQRPT HMNQMVFHKI 541 RNEDLIFNES LGQGTFTKIF KGVRREVGDY GQLHETEVLLKVLDKAHRNY SESFFEAASM 601 MSKLSHKHLV LNYGVCVCGD ENILVQEFVK FGSLDTYLKKNKNCINILWK LEVAKQLAWA 661 MHFLEENTLI HGNVCAKNIL LIREEDRKTG NPPFIKLSDPGISITVLPKD ILQERIPWVP 721 PECIENPKNL NLATDKWSFG TTLWEICSGG DKPLSALDSQRKLQFYEDRH QLPAPKWAEL 781 ANLINNCMDY EPDFRPSFRA IIRDLNSLFT PDYELLTENDMLPNMRIGAL GFSGAFEDRD 841 PTQFEERHLK FLQQLGKGNF GSVEMCRYDP LQDNTGEVVAVKKLQHSTEE HLRDFEREIE 901 ILKSLQHDNI VKYKGVCYSA GRRNLKLIME YLPYGSLRDYLQKHKERIDH IKLLQYTSQI 961 CKGMEYLGTK RYIHRDLATR NILVENENRV KIGDFGLTKVLPQDKEYYKV KEPGESPIFW 1021 YAPESLTESK FSVASDVWSF GVVLYELFTY IEKSKSPPAEFMRMIGNDKQ GQMIVFHLIE 1081 LLKNNGRLPR PDGCPDEIYM IMTECWNNNV NQRPSFRDLALRVDQIRDNM AG SEQ ID NO: 4 is the human wild type nucleotide sequencecorresponding to Jak2 (nucleotides 1-5285), wherein the underscoredbolded “ATG” denotes the beginning of the open reading frame: 1ctgcaggaag gagagaggaa gaggagcaga agggggcagc agcggacgcc gctaacggcc 61tccctcggcg ctgacaggct gggccggcgc ccggctcgct tgggtgttcg cgtcgccact 121tcggcttctc ggccggtcgg gcccctcggc ccgggcttgc ggcgcgcgtc ggggctgagg 181gctgctgcgg cgcagggaga ggcctggtcc tcgctgccga gggatgtgag tgggagctga 241gcccacactg gagggccccc gagggcccag cctggaggtc gttcagagcc gtgcccgtcc 301cggggcttcg cagaccttga cccgccgggt aggagccgcc cctgcgggct cgagggcgcg 361ctctggtcgc ccgatctgtg tagccggttt cagaagcagg caacaggaac aagatgtgaa 421ctgtttctct tctgcagaaa aagaggctct tcctcctcct cccgcgacgg caaatgttct 481gaaaaagact ctgc atg gga atggcctgcc ttacgatgac agaaatggag ggaacatcca 541cctcttctat atatcagaat ggtgatattt ctggaaatgc caattctatg aagcaaatag 601atccagttct tcaggtgtat ctttaccatt cccttgggaa atctgaggca gattatctga 661cctttccatc tggggagtat gttgcagaag aaatctgtat tgctgcttct aaagcttgtg 721gtatcacacc tgtgtatcat aatatgtttg ctttaatgag tgaaacagaa aggatctggt 781atccacccaa ccatgtcttc catatagatg agtcaaccag gcataatgta ctctacagaa 841taagatttta ctttcctcgt tggtattgca gtggcagcaa cagagcctat cggcatggaa 901tatctcgagg tgctgaagct cctcttcttg atgactttgt catgtcttac ctctttgctc 961agtggcggca tgattttgtg cacggatgga taaaagtacc tgtgactcat gaaacacagg 1021aagaatgtct tgggatggca gtgttagata tgatgagaat agccaaagaa aacgatcaaa 1081ccccactggc catctataac tctatcagct acaagacatt cttaccaaaa tgtattcgag 1141caaagatcca agactatcat attttgacaa ggaagcgaat aaggtacaga tttcgcagat 1201ttattcagca attcagccaa tgcaaagcca ctgccagaaa cttgaaactt aagtatctta 1261taaatctgga aactctgcag tctgccttct acacagagaa atttgaagta aaagaacctg 1321gaagtggtcc ttcaggtgag gagatttttg caaccattat aataactgga aacggtggaa 1381ttcagtggtc aagagggaaa cataaagaaa gtgagacact gacagaacag gatttacagt 1441tatattgcga ttttcctaat attattgatg tcagtattaa gcaagcaaac caagagggtt 1501caaatgaaag ccgagttgta actatccata agcaagatgg taaaaatctg gaaattgaac 1561ttagctcatt aagggaagct ttgtctttcg tgtcattaat tgatggatat tatagattaa 1621ctgcagatgc acatcattac ctctgtaaag aagtagcacc tccagccgtg cttgaaaata 1681tacaaagcaa ctgtcatggc ccaatttcga tggattttgc cattagtaaa ctgaagaaag 1741caggtaatca gactggactg tatgtacttc gatgcagtcc taaggacttt aataaatatt 1801ttttgacttt tgctgtcgag cgagaaaatg tcattgaata taaacactgt ttgattacaa 1861aaaatgagaa tgaagagtac aacctcagtg ggacaaagaa gaacttcagc agtcttaaag 1921atcttttgaa ttgttaccag atggaaactg ttcgctcaga caatataatt ttccagttta 1981ctaaatgctg tcccccaaag ccaaaagata aatcaaacct tctagtcttc agaacgaatg 2041gtgtttctga tgtaccaacc tcaccaacat tacagaggcc tactcatatg aaccaaatgg 2101tgtttcacaa aatcagaaat gaagatttga tatttaatga aagccttggc caaggcactt 2161ttacaaagat ttttaaaggc gtacgaagag aagtaggaga ctacggtcaa ctgcatgaaa 2221cagaagttct tttaaaagtt ctggataaag cacacagaaa ctattcagag tctttctttg 2281aagcagcaag tatgatgagc aagctttctc acaagcattt ggttttaaat tatggagtat 2341gtgtctgtgg agacgagaat attctggttc aggagtttgt aaaatttgga tcactagata 2401catatctgaa aaagaataaa aattgtataa atatattatg gaaacttgaa gttgctaaac 2461agttggcatg ggccatgcat tttctagaag aaaacaccct tattcatggg aatgtatgtg 2521ccaaaaatat tctgcttatc agagaagaag acaggaagac aggaaatcct cctttcatca 2581aacttagtga tcctggcatt agtattacag ttttgccaaa ggacattctt caggagagaa 2641taccatgggt accacctgaa tgcattgaaa atcctaaaaa tttaaatttg gcaacagaca 2701aatggagttt tggtaccact ttgtgggaaa tctgcagtgg aggagataaa cctctaagtg 2761ctctggattc tcaaagaaag ctacaatttt atgaagatag gcatcagctt cctgcaccaa 2821agtgggcaga attagcaaac cttataaata attgtatgga ttatgaacca gatttcaggc 2881cttctttcag agccatcata cgagatctta acagtttgtt tactccagat tatgaactat 2941taacagaaaa tgacatgtta ccaaatatga ggataggtgc cctggggttt tctggtgcct 3001ttgaagaccg ggatcctaca cagtttgaag agagacattt gaaatttcta cagcaacttg 3061gcaagggtaa ttttgggagt gtggagatgt gccggtatga ccctctacag gacaacactg 3121gggaggtggt cgctgtaaaa aagcttcagc atagtactga agagcaccta agagactttg 3181aaagggaaat tgaaatcctg aaatccctac agcatgacaa cattgtaaag tacaagggag 3241tgtgctacag tgctggtcgg cgtaatctaa aattaattat ggaatattta ccatatggaa 3301gtttacgaga ctatcttcaa aaacataaag aacggataga tcacataaaa cttctgcagt 3361acacatctca gatatgcaag ggtatggagt atcttggtac aaaaaggtat atccacaggg 3421atctggcaac gagaaatata ttggtggaga acgagaacag agttaaaatt ggagattttg 3481ggttaaccaa agtcttgcca caagacaaag aatactataa agtaaaagaa cctggtgaaa 3541gtcccatatt ctggtatgct ccagaatcac tgacagagag caagttttct gtggcctcag 3601atgtttggag ctttggagtg gttctgtatg aacttttcac atacattgag aagagtaaaa 3661gtccaccagc ggaatttatg cgtatgattg gcaatgacaa acaaggacag atgatcgtgt 3721tccatttgat agaacttttg aagaataatg gaagattacc aagaccagat ggatgcccag 3781atgagatcta tatgatcatg acagaatgct ggaacaataa tgtaaatcaa cgcccctcct 3841ttagggatct agctcttcga gtggatcaaa taagggataa catggctgga tgaaagaaat 3901gaccttcatt ctgagaccaa agtagattta cagaacaaag ttttatattt cacattgctg 3961tggactatta ttacatatat cattattata taaatcatga tgctagccag caaagatgtg 4021aaaatatctg ctcaaaactt tcaaagttta gtaagttttt cttcatgagg ccaccagtaa 4081aagacattaa tgagaattcc ttagcaagga ttttgtaaga agtttcttaa acattgtcag 4141ttaacatcac tcttgtctgg caaaagaaaa aaaatagact ttttcaactc agctttttga 4201gacctgaaaa aattattatg taaattttgc aatgttaaag atgcacagaa tatgtatgta 4261tagtttttac cacagtggat gtataatacc ttggcatctt gtgtgatgtt ttacacacat 4321gagggctggt gttcattaat actgttttct aatttttcca tagttaatct ataattaatt 4381acttcactat acaaacaaat taagatgttc agataattga ataagtacct ttgtgtcctt 4441gttcatttat atcgctggcc agcattataa gcaggtgtat acttttagct tgtagttcca 4501tgtactgtaa atatttttca cataaaggga acaaatgtct agttttattt gtataggaaa 4561tttccctgac cctaaataat acattttgaa atgaaacaag cttacaaaga tataatctat 4621tttattatgg tttcccttgt atctatttgt ggtgaatgtg ttttttaaat ggaactatct 4681ccaaattttt ctaagactac tatgaacagt tttcttttaa aattttgaga ttaagaatgc 4741caggaatatt gtcatccttt gagctgctga ctgccaataa cattcttcga tctctgggat 4801ttatgctcat gaactaaatt taagcttaag ccataaaata gattagattg ttttttaaaa 4861atggatagct cattaagaag tgcagcaggt taagaatttt ttcctaaaga ctgtatattt 4921gaggggtttc agaattttgc attgcagtca tagaagagat ttatttcctt tttagagggg 4981aaatgaggta aataagtaaa aaagtatgct tgttaatttt attcaagaat gccagtagaa 5041aattcataac gtgtatcttt aagaaaaatg agcatacatc ttaaatcttt tcaattaagt 5101ataaggggtt gttcgttgtt gtcatttgtt atagtgctac tccactttag acaccatagc 5161taaaataaaa tatggtgggt tttgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 5221tgttatttat acaaaactta aaatacttgc tgttttgatt aaaaagaaaa tagtttctta 5281cttta

The polypeptide sequence of human Jak 3 is depicted in SEQ ID NO: 109.The nucleotide sequence of human Jak 3 is shown in SEQ ID NO: 110.Sequence information related to Jak 3 is accessible in public databasesby GenBank Accession numbers NP_000206 (for protein) and NM_000215 (fornucleic acid). JAK3 is a downstream signaling partner of the IL-2receptor common gamma chain, which is shared with the IL-2, -4, -7, -9,-15, and -21 receptors.

SEQ ID NO: 109 is the human wild type amino acid sequence correspondingto Jak3 (residues 1-1124): 1 MAPPSEETPL IPQRSCSLLS TEAGALHVLL PARGPGPPQRLSFSFGDHLA EDLCVQAAKA 61 SGILPVYHSL FALATEDLSC WFPPSHIFSV EDASTQVLLYRIRFYFPNWF GLEKCHRFGL 121 RKDLASAILD LPVLEHLFAQ HRSDLVSGRL PVGLSLKEQGECLSLAVLDL ARMAREQAQR 181 PGELLKTVSY KACLPPSLRD LIQGLSFVTR RRIRRTVRRALRRVAACQAD RHSLMAKYIM 241 DLERLDPAGA AETFHVGLPG ALGGHDGLGL LRVAGDGGIAWTQGEQEVLQ PFCDFPEIVD 301 ISIKQAPRVG PAGEHRLVTV TRTDNQILEA EFPGLPEALSFVALVDGYFR LTTDSQHFFC 361 KEVAPPRLLE EVAEQCHGPI TLDFAINKLK TGGSRPGSYVLRRSPQDFDS FLLTVCVQNP 421 LGPDYKGCLI RRSPTGTFLL VGLSRPHSSL RELLATCWDGGLHVDGVAVT LTSCCIPRPK 481 EKSNLIVVQR GHSPPTSSLV QPQSQYQLSQ MTFHKIPADSLEWHENLGHG SFTKIYRGCR 541 HEVVDGEARK TEVLLKVMDA KHKNCMESFL EAASLMSQVSYRHLVLLHGV CMAGDSTMVQ 601 EFVHLGAIDM YLRKRGHLVP ASWKLQVVKQ LAYALNYLEDKGLPHGNVSA RKVLLAREGA 661 DGSPPFIKLS DPGVSPAVLS LEMLTDRIPW VAPECLREAQTLSLEADKWG FGATVWEVFS 721 GVTMPISALD PAKKLQFYED RQQLPAPKWT ELALLIQQCMAYEPVQRPSF RAVIRDLNSL 781 ISSDYELLSD PTPGALAPRD GLWNGAQLYA CQDPTIFEERHLKYISQLGK GNFGSVELCR 841 YDPLGDNTGA LVAVKQLQHS GPDQQRDFQR EIQILKALHSDFIVKYRGVS YGPGRQSLRL 901 VMEYLPSGCL RDFLQRHRAR LDASRLLLYS SQICKGMEYLGSRRCVHRDL AARNILVESE 961 AHVKIADFGL AKLLPLDKDY YVVREPGQSP IFWYAPESLSDNIFSRQSDV WSFGVVLYEL 1021 FTYCDKSCSP SAEFLRMMGC ERDVPALCRL LELLEEGQRLPAPPACPAEV HELMKLCWAP 1081 SPQDRPSFSA LGPQLDMLWS GSRGCETHAF TAHPEGKHHSLSFS SEQ ID NO: 110 is the human wild type nucleotide sequencecorresponding to Jak3 (nucleotides 1-5449), wherein the underscoredbolded “ATG” denotes the beginning of the open reading frame: 1cacacaggaa ggagccgagt gggactttcc tctcgctgcc tcccggctct gcccgccctt 61cgaaagtcca gggtccctgc ccgctaggca agttgcactc atg gcacctc caagtgaaga 121gacgcccctg atccctcagc gttcatgcag cctcttgtcc acggaggctg gtgccctgca 181tgtgctgctg cccgctcggg gccccgggcc cccccagcgc ctatctttct cctttgggga 241ccacttggct gaggacctgt gcgtgcaggc tgccaaggcc agcggcatcc tgcctgtgta 301ccactccctc tttgctctgg ccacggagga cctgtcctgc tggttccccc cgagccacat 361cttctccgtg gaggatgcca gcacccaagt cctgctgtac aggattcgct tttacttccc 421caattggttt gggctggaga agtgccaccg cttcgggcta cgcaaggatt tggccagtgc 481tatccttgac ctgccagtcc tggagcacct ctttgcccag caccgcagtg acctggtgag 541tgggcgcctc cccgtgggcc tcagtctcaa ggagcagggt gagtgtctca gcctggccgt 601gttggacctg gcccggatgg cgcgagagca ggcccagcgg ccgggagagc tgctgaagac 661tgtcagctac aaggcctgcc tacccccaag cctgcgcgac ctgatccagg gcctgagctt 721cgtgacgcgg aggcgtattc ggaggacggt gcgcagagcc ctgcgccgcg tggccgcctg 781ccaggcagac cggcactcgc tcatggccaa gtacatcatg gacctggagc ggctggatcc 841agccggggcc gccgagacct tccacgtggg cctccctggg gcccttggtg gccacgacgg 901gctggggctg ctccgcgtgg ctggtgacgg cggcatcgcc tggacccagg gagaacagga 961ggtcctccag cccttctgcg actttccaga aatcgtagac attagcatca agcaggcccc 1021gcgcgttggc ccggccggag agcaccgcct ggtcactgtt accaggacag acaaccagat 1081tttagaggcc gagttcccag ggctgcccga ggctctgtcg ttcgtggcgc tcgtggacgg 1141ctacttccgg ctgaccacgg actcccagca cttcttctgc aaggaggtgg caccgccgag 1201gctgctggag gaagtggccg agcagtgcca cggccccatc actctggact ttgccatcaa 1261caagctcaag actgggggct cacgtcctgg ctcctatgtt ctccgccgca gcccccagga 1321ctttgacagc ttcctcctca ctgtctgtgt ccagaacccc cttggtcctg attataaggg 1381ctgcctcatc cggcgcagcc ccacaggaac cttccttctg gttggcctca gccgacccca 1441cagcagtctt cgagagctcc tggcaacctg ctgggatggg gggctgcacg tagatggggt 1501ggcagtgacc ctcacttcct gctgtatccc cagacccaaa gaaaagtcca acctgatcgt 1561ggtccagaga ggtcacagcc cacccacatc atccttggtt cagccccaat cccaatacca 1621gctgagtcag atgacatttc acaagatccc tgctgacagc ctggagtggc atgagaacct 1681gggccatggg tccttcacca agatttaccg gggctgtcgc catgaggtgg tggatgggga 1741ggcccgaaag acagaggtgc tgctgaaggt catggatgcc aagcacaaga actgcatgga 1801gtcattcctg gaagcagcga gcttgatgag ccaagtgtcg taccggcatc tcgtgctgct 1861ccacggcgtg tgcatggctg gagacagcac catggtgcag gaatttgtac acctgggggc 1921catagacatg tatctgcgaa aacgtggcca cctggtgcca gccagctgga agctgcaggt 1981ggtcaaacag ctggcctacg ccctcaacta tctggaggac aaaggcctgc cccatggcaa 2041tgtctctgcc cggaaggtgc tcctggctcg ggagggggct gatgggagcc cgcccttcat 2101caagctgagt gaccctgggg tcagccccgc tgtgttaagc ctggagatgc tcaccgacag 2161gatcccctgg gtggcccccg agtgtctccg ggaggcgcag acacttagct tggaagctga 2221caagtggggc ttcggcgcca cggtctggga agtgtttagt ggcgtcacca tgcccatcag 2281tgccctggat cctgctaaga aactccaatt ttatgaggac cggcagcagc tgccggcccc 2341caagtggaca gagctggccc tgctgattca acagtgcatg gcctatgagc cggtccagag 2401gccctccttc cgagccgtca ttcgtgacct caatagcctc atctcttcag actatgagct 2461cctctcagac cccacacctg gtgccctggc acctcgtgat gggctgtgga atggtgccca 2521gctctatgcc tgccaagacc ccacgatctt cgaggagaga cacctcaagt acatctcaca 2581gctgggcaag ggcaactttg gcagcgtgga gctgtgccgc tatgacccgc taggcgacaa 2641tacaggtgcc ctggtggccg tgaaacagct gcagcacagc gggccagacc agcagaggga 2701ctttcagcgg gagattcaga tcctcaaagc actgcacagt gatttcattg tcaagtatcg 2761tggtgtcagc tatggcccgg gccgccagag cctgcggctg gtcatggagt acctgcccag 2821cggctgcttg cgcgacttcc tgcagcggca ccgcgcgcgc ctcgatgcca gccgcctcct 2881tctctattcc tcgcagatct gcaagggcat ggagtacctg ggctcccgcc gctgcgtgca 2941ccgcgacctg gccgcccgaa acatcctcgt ggagagcgag gcacacgtca agatcgctga 3001cttcggccta gctaagctgc tgccgcttga caaagactac tacgtggtcc gcgagccagg 3061ccagagcccc attttctggt atgcccccga atccctctcg gacaacatct tctctcgcca 3121gtcagacgtc tggagcttcg gggtcgtcct gtacgagctc ttcacctact gcgacaaaag 3181ctgcagcccc tcggccgagt tcctgcggat gatgggatgt gagcgggatg tccccgccct 3241ctgccgcctc ttggaactgc tggaggaggg ccagaggctg ccggcgcctc ctgcctgccc 3301tgctgaggtt cacgagctca tgaagctgtg ctgggcccct agcccacagg accggccatc 3361attcagcgcc ctgggccccc agctggacat gctgtggagc ggaagccggg ggtgtgagac 3421tcatgccttc actgctcacc cagagggcaa acaccactcc ctgtcctttt catagctcct 3481gcccgcagac ctctggatta ggtctctgtt gactggctgt gtgaccttag gcccggagct 3541gcccctctct gggcctcaga ggccttatga gggtcctcta cttcaggaac acccccatga 3601cattgcattt gggggggctc ccgtggcctg tagaatagcc tgtggccttt gcaatttgtt 3661aaggttcaag acagatgggc atatgtgtca gtggggctct ctgagtcctg gcccaaagaa 3721gcaaggaacc aaatttaaga ctctcgcatc ttcccaaccc cttaagccct ggccccctga 3781gtttcctttt ctgtctctct ctttttattt tttttatttt tatttttatt tttgagacag 3841agcctcgctc tgttacccag ggtggagtgc agtggtgcga tctcggctca gtgcaacctc 3901tgcttcccag gttcaagcga ttctcctgcc tcagcctccc gagtagctgg gattacaggt 3961gtgcaccacc acacccggct aatttttttt atttttaata gagatgaggt ttcaccatga 4021tggccaggct gatctcgaac tcctaacctc aagtgatcct cccacctcag cctcccaaag 4081tgttggaata ataggcatga gccactgcac ccaggctttt ttttttttaa atttattatt 4141attattttta agagacagga tcttgctacg ttgcccaggc tggtcttgaa ctcctgggct 4201acagtgatcc tcctgcctta tcctcctaaa tagctgggac tacagcacct agttttgagt 4261ttcctgtctt atttccaatg gggacattca tgtagctttt tttttttttt tttttttgag 4321acggagtctc gctctgtcgc ccaggctgga gtacagtggc gcaatctagg ctcactgcaa 4381gctccgcctc ctgggttcac accattctct cgcctcagcc tcccaagtag ctgggactac 4441aggcgcccgc caccacaccc ggctaatttt ttgtattttt agtagagacg gggtttcacc 4501ttgttagcca ggatggtttc catctcctga cctcgtgatc tgcccgtctc ggcctcccaa 4561agtgctggga ttacaggcat gagccactgc gcccggccct catgtagctt taaatgtatg 4621atctgacttc tgctccccga tctctgtttc tctggaggaa gccaaggaca agagcagttg 4681ctgtggctgg gactctgcct tttaggggag cccgtgtatc tctttgggat cctgaaaggg 4741ggcaggaaag gctggggtcc cagtccaccc taatggtatc tgagtgtcct agggcttcag 4801ttttcccacc tgtccaatgg gaccctttct gtcctcaccc tacaaggggc acaaagggat 4861gacaccaaac ctggcaggaa cttttcacgc aatcaaggga aggaaaggca ttcctggcag 4921agggaacagc atgccaagcg tgagaaggct cagagtaagg aggttaagag cccaagtatt 4981ggagcctaca gttttgcccc ttccatgcag tgtgacagtg ggcaagttcc tttccctctc 5041tgggtctcag ttctgtcccc tgcaaaatgg tcagagctta ccccttggct gtgcagggtc 5101aactttctga ctggtgagag ggattctcat gcaggttaag cttctgctgc tcctcctcac 5161ctgcaaagct tttctgccac ttttgcctcc ttggaaaact cttatccatc tctcaaaact 5221ccagctacca catccttgca gccttccctc atataccccc actactactg tagccctgtc 5281cttccctcca gccccactct ggccctgggg ctggggaagt gtctgtgtcc agctgtctcc 5341cctgacctca gggttccttg ggggctgggc tgaggcctca gtacagaggg ggctctggaa 5401atgtttgttg actgaataaa ggaattcagt ggaaaaaaaa aaaaaaaaa

The polypeptide sequence of human Stat 1 is depicted in SEQ ID NO: 5.The nucleotide sequence of human Stat 1 is shown in SEQ ID NO: 6.Sequence information related to Stat 1 is accessible in public databasesby GenBank Accession numbers ADA59516 (for protein) and GU211347.1 (fornucleic acid).

SEQ ID NO: 5 is the human wild type amino acid sequence corresponding toStat1 (residues 1-750): 1 MSQWYELQQL DSKFLEQVHQ LYDDSFPMEI RQYLAQWLEKQDWEHTANDV SFATIRFHDL 61 LSQLDDQYSR FSLENNFLLQ HNIRKSKRNL QDNFQEDPIQMSMIIYSCLK EERKILENAQ 121 RFNQAQSGNI QSTVMLDKQK ELDSKVRNVK DKVMCIEHEIKSLEDLQDEY DFKCKTLQNR 181 EHETNGVAKS DQKQEQLLLK KMYLMLDNKR KEVVHKIIELLNVTELTQNA LINDELVEWK 241 RRQQSACIGG PPNACLDQLQ NWFTIVAESL QQVRQQLKKLEELEQKYTYE HDPITKNKQV 301 LWDRTFSLFQ QLIQSSFVVE RQPCMPTHPQ RPLVLKTGVQFTVKLRLLVK LQELNYNLKV 361 KVLFDKDVNE RNTVKGFRKF NILGTHTKVM NMEESTNGSLAAEFRHLQLK EQKNAGTRTN 421 EGPLIVTEEL HSLSFETQLC QPGLVIDLET TSLPVVVISNVSQLPSGWAS ILWYNMLVAE 481 PRNLSFFLTP PCARWAQLSE VLSWQFSSVT KRGLNVDQLNMLGEKLLGPN ASPDGLIPWT 541 RFCKENINDK NFPFWLWIES ILELIKKHLL PLWNDGCIMGFISKERERAL LKDQQPGTFL 601 LRFSESSREG AITFTWVERS QNGGEPDFHA VEPYTKKELSAVTFPDIIRN YKVMAAENIP 661 ENPLKYLYPN IDKDHAFGKY YSRPKEAPEP MELDGPKGTGYIKTELISVS EVHPSRLQTT 721 DNLLPMSPEE FDEVSRIVGS VEFDSMMNTV SEQ ID NO: 6is the human wild type nucleotide sequence corresponding to Stat1(nucleotides 1-2353), wherein the underscored bolded “ATG” denotes thebeginning of the open reading frame: 1 gtgctgtgcg tagctgctcc tttggttgaatccccaggcc cttgttgggg cacaaggtgg 61 cagg atg tct cagtggtacg aacttcagcagcttgactca aaattcctgg agcaggttca 121 ccagctttat gatgacagtt ttcccatggaaatcagacag tacctggcac agtggttaga 181 aaagcaagac tgggagcaca ctgccaatgatgtttcattt gccaccatcc gttttcatga 241 cctcctgtca cagctggatg atcaatatagtcgcttttct ttggagaata acttcttgct 301 acagcataac ataaggaaaa gcaagcgtaatcttcaggat aattttcagg aagacccaat 361 ccagatgtct atgatcattt acagctgtctgaaggaagaa aggaaaattc tggaaaacgc 421 ccagagattt aatcaggctc agtcggggaatattcagagc acagtgatgt tagacaaaca 481 gaaagagctt gacagtaaag tcagaaatgtgaaggacaag gttatgtgta tagagcatga 541 aatcaagagc ctggaagatt tacaagatgaatatgacttc aaatgcaaaa ccttgcagaa 601 cagagaacac gagaccaatg gtgtggcaaagagtgatcag aaacaagaac agctgttact 661 caagaagatg tatttaatgc ttgacaataagagaaaggaa gtagttcaca aaataataga 721 gttgctgaat gtcactgaac ttacccagaatgccctgatt aatgatgaac tagtggagtg 781 gaagcggaga cagcagagcg cctgtattggggggccgccc aatgcttgct tggatcagct 841 gcagaactgg ttcactatag ttgcggagagtctgcagcaa gttcggcagc agcttaaaaa 901 gttggaggaa ttggaacaga aatacacctacgaacatgac cctatcacaa aaaacaaaca 961 agtgttatgg gaccgcacct tcagtcttttccagcagctc attcagagct cgtttgtggt 1021 ggaaagacag ccctgcatgc caacgcaccctcagaggccg ctggtcttga agacaggggt 1081 ccagttcact gtgaagttga gactgttggtgaaattgcaa gagctgaatt ataatttgaa 1141 agtcaaagtc ttatttgata aagatgtgaatgagagaaat acagtaaaag gatttaggaa 1201 gttcaacatt ttgggcacgc acacaaaagtgatgaacatg gaggagtcca ccaatggcag 1261 tctggcggct gaatttcggc acctgcaattgaaagaacag aaaaatgctg gcaccagaac 1321 gaatgagggt cctctcatcg ttactgaagagcttcactcc cttagttttg aaacccaatt 1381 gtgccagcct ggtttggtaa ttgacctcgagacgacctct ctgcccgttg tggtgatctc 1441 caacgtcagc cagctcccga gcggttgggcctccatcctt tggtacaaca tgctggtggc 1501 ggaacccagg aatctgtcct tcttcctgactccaccatgt gcacgatggg ctcagctttc 1561 agaagtgctg agttggcagt tttcttctgtcaccaaaaga ggtctcaatg tggaccagct 1621 gaacatgttg ggagagaagc ttcttggtcctaacgccagc cccgatggtc tcattccgtg 1681 gacgaggttt tgtaaggaaa atataaatgataaaaatttt cccttctggc tttggattga 1741 aagcatccta gaactcatta aaaaacacctgctccctctc tggaatgatg ggtgcatcat 1801 gggcttcatc agcaaggagc gagagcgtgccctgttgaag gaccagcagc cggggacctt 1861 cctgctgcgg ttcagtgaga gctcccgggaaggggccatc acattcacat gggtggagcg 1921 gtcccagaac ggaggcgaac ctgacttccatgcggttgaa ccctacacga agaaagaact 1981 ttctgctgtt actttccctg acatcattcgcaattacaaa gtcatggctg ctgagaatat 2041 tcctgagaat cccctgaagt atctgtatccaaatattgac aaagaccatg cctttggaaa 2101 gtattactcc aggccaaagg aagcaccagagccaatggaa cttgatggcc ctaaaggaac 2161 tggatatatc aagactgagt tgatttctgtgtctgaagtt cacccttcta gacttcagac 2221 cacagacaac ctgctcccca tgtctcctgaggagtttgac gaggtgtctc ggatagtggg 2281 ctctgtagaa ttcgacagta tgatgaacacagtatagagc atgaattttt ttcatcttct 2341 ctggcgacag ttt

The polypeptide sequence of human Stat 2 is depicted in SEQ ID NO: 7.The nucleotide sequence of human Stat 2 is shown in SEQ ID NO: 8.Sequence information related to Stat 2 is accessible in public databasesby GenBank Accession numbers AAA98760 (for protein) and U18671.1 (fornucleic acid).

SEQ ID NO: 7 is the human wild type amino acid sequence corresponding toStat2 (residues 1-851): 1 MAQWEMLQNL DSPFQDQLHQ LYSHSLLPVD IRQYLAVWIEDQNWQEAALG SDDSKATMLF 61 FHFLDQLNYE CGRCSQDPES LLLQHNLRKF CRDIQPFSQDPTQLAEMIFN LLLEEKRILI 121 QAQRAQLEQG EPVLETPVES QQHEIESRIL DLRAMMEKLVKSISQLKDQQ DVFCFRYKIQ 181 AKGKTPSLDP HQTKEQKILQ ETLNELDKRR KEVLDASKALLGRLTTLIEL LLPKLEEWKA 241 QQQKACIRAP IDHGLEQLET WFTAGAKLLF HLRQLLKELKGLSCLVSYQD DPLTKGVDLR 301 NAQVTELLQR LLHRAFVVET QPCMPQTPHR PLILKTGSKFTVRTRLLVRL QEGNESLTVE 361 VSIDRNPPQL QGFRKFNILT SNQKTLTPEK GQSQGLIWDFGYLTLVEQRS GGSGKGSNKG 421 PLGVTEELHI ISFTVKYTYQ GLKQELKTDT LPVVIISNMNQLSIAWASVL WFNLLSPNLQ 481 NQQFFSNPPK APWSLLGPAL SWQFSSYVGR GLNSDQLSMLRNKLFGQNCR TEDPLLSWAD 541 FTKRESPPGK LPFWTWLDKI LELVHDHLKD LWNDGRIMGFVSRSQERRLL KKTMSGTFLL 601 RFSESSEGGI TCSWVEHQDD DKVLIYSVQP YTKEVLQSLPLTEIIRHYQL LTEENIPENP 661 LRFLYPRIPR DEAFGCYYQE KVNLQERRKY LKHRLIVVSNRQVDELQQPL ELKPEPELES 721 LELELGLVPE PELSLDLEPL LKAGLDLGPE LESVLESTLEPVIEPTLCMV SQTVPEPDQG 781 PVSQPVPEPD LPCDLRHLNT EPMEIFRNCV KIEEIMPNGDPLLAGQNTVD EVYVSRPSHF 841 YTDGPLMPSD F SEQ ID NO: 8 is the human wildtype nucleotide sequence corresponding to Stat2 (nucleotides 1-18648),wherein the underscored bolded “ATG” denotes the beginning of the openreading frame: 1 tcaagatcag cctgggcaac atggcgaaac cccgtctcta caataaatacaaaaaaatta 61 tcctggcgga gttatgcacg ttgtagtccc aactacctgg gaggctgaggcgggagaatc 121 acctgagcct gggaggtcga ggctgcagcg agccgagatc ggccgctgcattccagcctg 181 ggtgacagag cgagaccatg tctcaaaaaa taaaaattaa aaaaaaattgttttcattac 241 ctcagccctc ctcttcctat cccaaggcgt cgaaattccg gtcccaccccttcccatgga 301 gcccttggcg tctccaggct cctcaagcta gtttcggttc cgggctcacgcgcgggttct 361 cgaaaatcag ctgtttcagt cttgggctag tccactaatt ggactcctcccctcgtagaa 421 agtgcctact tgaacttctc caccaatcgc tgaagctgca ggtgtggtttcggctcagct 481 tgtcccgccc tggcggaggg gcggagttgc ggcggcgcca gtgagctcgcagtctgggaa 541 gggcttgact gaatggcagc cagtgtcggg gtggcggctg ggaatgggggccgctccgga 601 cttccgctgc caactacaag ggggcgggtc cgaggggggt tagccgaagttgtaggcggg 661 gcgcgaggtt ctagtacccg agctcatact agggacggga agtcgcgaccagagccattg 721 gagggcgcgg ggactgcaac cctaatcagg tacgggccct gagagggtgtgctggggtag 781 gggtgggggt gagagtgaga gttcctccga gggaagggcg actggcccaggggttacccc 841 ctggagaggg tagcttcctt ccccagattg aaataggagc tgtcgcctgctcggtcctcg 901 atcttcttct gtccagccta tctccctaac cctaatgccc ctctcccaaaactgccctgc 961 agcttccgag acccggaatc tggcattgtt atgttggttc ggtatctgacgtttttccct 1021 ctgctctgca ttatttttta tcttcaccaa aaaacgatgt tcaaagatagataaatctaa 1081 aaacaaagat agataaatct attacccttg tttcgtaaaa agtataagctactgaaagat 1141 gaaacgattg cctaaggtca cacacaaaat tcagttcatt tcagaaaagcttcttgagtg 1201 caaaatatgt gcctaagaat gagagataat gagaaaaaat tgtttcagccccttaacctc 1261 agtgtttgca atccatttgg ggagaccagg ttttttgttt ttgttttcatatttgaatct 1321 ttgctgactt gctcctttaa tatcagacac ttaaatcctc agatgggactcatcatattt 1381 tttttgagat ggaatcttca ctatgttgct caagcttggt ctgcaactcctggctcaagc 1441 catcctctcg tcttgttggg cctctcgtct tgtgggcctg cacaaagtgctgggattaca 1501 ggcatgagcc attcatgccc tgggcgcacc ttggattgcg atgtgtgtgtgttgtgaagc 1561 tttttttttt ggtatcataa aagcaataca gatacatagt tttaaaaatcaagcagctac 1621 taaaagagtt aaaatgaaaa tagcccctcc caatccctcc cttgttcctgctggaggtag 1681 aaaggcagct gatgttattc atgttagtag aagactctcc caccccaagcatttctcttt 1741 attttgtaat aaaatcatgt gaccttttta gaccacaaat atgcatgaattctgttctgt 1801 taggctcagg ctgcaacaag ataagtttca gtttcctaaa tagacaccagctggcagtga 1861 gcagggaaca gtggggagaa agatgcatgg gacagcctgc ttggtgacaggcaaaaaccg 1921 gtttgttgtt cttttagaga cagagtcttg ctttgtcacc caggctggagtgtagtgatg 1981 tgatctctgc ttactgcaac cctgcctctg ggtacaagcc attctcctgcctcagcctct 2041 tgagtagctg ggattacagg caacaatttt aagtgaagtg aagtttcaggatctcgagca 2101 aagttgtata acctataatc atattcaaga ttcacaggtc ataaacgtgtcatattcttg 2161 ggattgagcg acccattgca cagcatttag atgtgcttct agaatggagctcctccttcc 2221 tatatggagg gcagtttata tggtgtactt acctgaccac caaaaagatttggctctaaa 2281 aaagcttcag gtggccgggc atggtggttc acccctgtaa tccagcactttgggaggcag 2341 gtgggcagat cacctgaggt cagaagttca gacagctgga catatggtgaaacctcatct 2401 ctactaaaaa tacaaaaatt agactgggca tggtagtggg cgcctgtaatcccagctagt 2461 cgggaggctg aggcaggaga atcccttcaa ctcggacggc agagtttgcagtgaggccga 2521 gatcgtgtca ctgcagtcca gcctgggtga cagagcaaga ctccatctcaaaaaaagtaa 2581 aaaaaaaaaa aagaaaaaaa aaagcttcag agccagcagg gatcatgctgtaataaatac 2641 ttaacatcaa cactgatctt taaatgcttt agcacaatca aatataaataacaaacacac 2701 acataaatgc aaaataaatg aattagggag atagatgaaa taagattgtggaaatagtaa 2761 tgtttgttaa agctggatgg tgatccttgt actattcact ctactctagtgtgtatttga 2821 aaattaccat taggctggtt atggtggctc atgcctgtta atcccggcattttggaaggc 2881 tgaggcaggc ggattacttg agctcaggag tttagagtct gcctgggcaacatggcaaaa 2941 tcccatctct acaaaaaatt agctggcatg atggcacact cctgtagtcccagctccttg 3001 aggggctgag gcagagaatg gcttgaacct gagaggctaa agctgcagtgagccaagatc 3061 atgccactgc actccagcct gggtgaccaa gtgagaccct gtctcaaaaaaaaaaaaaaa 3121 aaaaagaaaa gaaaattccc attaaagcac aaaggcccac ttattgaagctattaaaata 3181 caggttgggg ccggctgggc atcgcgtcac gcctgtaatc ccagcactttggaaggccga 3241 ggtaggcgag tcacgagttc aggagatcga gaccatcctg gctaacacggtgaaacccca 3301 tctctactaa aaatacaaaa aaaaaaatca gccgggcatg gtggcgggagcctatagtcc 3361 cagctactcg ggaggctgag gcaggagaat ggcatgagcc cgggaggcggagcttgcagt 3421 gagccaaaat cacaccactg cactccagcc tgggcaacag atcgagactccatctgaaga 3481 aaaaaaaaat acaggttggg accacagtgg ctcatgcctg taatcctagtactttgggag 3541 tccgaagtag gtggatcacc tgaggtcagg actttgagac cagcctggccaacatggcaa 3601 aaccccatct ctactaaaaa atatacaaaa attagctggg cgtggtggtgggtgcctgta 3661 atcccagcta ctcaggaggc tgaggcagaa gaatcacaac aaccagggggatggtggttg 3721 caatgagcca agatcatctc cacttcactc cggcccaggc aaaagagtgagagtcatctt 3781 aaaaaaaaaa aaaaaaaaaa aaaaaaaata cagattaggc attcctaatctgaaaaattt 3841 ggctccaaaa tgctccagtc gagcatttcc tttgagtgtc atgtgggtgctcaaaaagtt 3901 agatttttgg accattttca gatttcagag ttttggatta gggatgctcgactggtaagt 3961 aatcgagata ttccaaaaat ctggacaaat ctgaaatcca aaatgcttggaatagcagat 4021 actcaactgg tagcactccc tggaagaata tgcaccaaac tgatagcagtggttaccttc 4081 tggtgaggag gggaaagaac caagattagc agtaggatca acatatattttaatgttttc 4141 tgtattttta ttacttgtat aatttaaaca ttttaaatta gtaataatgaacaatcatga 4201 aactatggat gatttagtcc agcaaaatat ccaattggga accctcatccttctgcagag 4261 cccaa atg gc gcagtgggaa atgctgcaga atcttgacag cccctttcaggatcagctgc 4321 accagcttta ctcgcacagc ctcctgcctg tggacattcg acagtacttggctgtctgga 4381 ttgaagacca gaactggtga ggccttcagg aagttggggg aatgaaaaaggtggccttcc 4441 acttctgggc ccccgggatc ctggaatcat taatggcagg aaggggttggaaagcctcag 4501 gactacagta acactgcaga gacactaata cttcttattc ctggtcccaggcaggaagct 4561 gcacttggga gtgatgattc caaggctacc atgctattct tccacttcttggatcagctg 4621 aactatgagt gtggccgttg cagccaggac ccagagtcct tgttgctgcagcacaatttg 4681 cggaaattct gccgggacat tcaggtactt ggaacggttg ggagtgatggggtagcactg 4741 ggagcagagc atagaggagt aaggtttgga gaatagaata gtacctggaggtggcaaggg 4801 agacgggaac aaatgtgggg aaaggaggac agagtctgga cttggggaatcactagcaga 4861 gagaagggtt gcatatacgt gacactgttg ggaggatgct atggtgaaaagacaaagggc 4921 taagaacccc gaaggaggag gaaatactgt ggacattggt ggggagggtctagggcaata 4981 ggtcattgag agtggttgaa ttggatcaat cctttctgtt tacctttctgttagcccttt 5041 tcccaggatc ctacccagtt ggctgagatg atctttaacc tccttctggaagaaaaaaga 5101 attttgatcc aggctcagag ggcccaattg gtgaggacaa ttcagtggtaatgttggaaa 5161 ctcctgaagt agagaggaac catggaaagg actcagggag ttgtctcagaacaggatccc 5221 cccgacatcc tgtggtataa tttcaggcct gaacttaagg catgaaaggccagagttaaa 5281 acgtgctcag agcctctttt ttcaggaaca aggagagcca gttctcgaaacacctgtgga 5341 gagccagcaa catgagattg aatcccggat cctggattta agggctatgatggaggttag 5401 tagatgtggt aggagttagg gttgacagtg ttcagcctaa cacctccctgagaagcagcc 5461 tcatcggggt cctctcccct ctgcagaagc tggtaaaatc catcagccaactgaaagacc 5521 agcaggatgt cttctgcttc cgatataaga tccaggccaa aggtaggaagcacattgagg 5581 ggctggagaa agataagtgc ctgctgagaa gccggagctg gaagtgaacaggagaaagct 5641 ccgatgagca gtagtcactg tcagacacac cccactgact acagtcctgctgccgtgcaa 5701 agctggaatc gtgctttgtg gaggctgagc tggaggtgac agctgagagacagtaaattg 5761 ttgaggaaat gcatggaaaa ctaacagtgt tttatttgag ggggtgtctggtccaagatg 5821 accacttcag aatttgcctg gagggtccca caggtgcctg tgctttgcttggtttccctt 5881 tcttcctccg ccacaaaatt cctccttcct gactctgact gagaccccagtcaggaagga 5941 gaggaaagaa cccctggact gactcctgtt cccaccatcc agggaagacaccctctctgg 6001 acccccatca gaccaaagag cagaagattc tgcaggaaac tctcaatgaactggacaaaa 6061 ggagaaaggt gggaggcagc agaacagaac atgtgggcaa caaggacctgaaaaaatgag 6121 ggatgttggg aaccctggta atctagcgct ggcttctttc tttcttcatccccagttggg 6181 tggtggaggg tgaaagggag agatgctcaa cactcacatt atctctttcccaggaggtgc 6241 tggatgcctc caaagcactg ctaggccgat taactaccct aatcgagctactgctgccaa 6301 agttggagga gtggaaggcc cagcagcaaa aagcctgcat cagagctcccattgaccacg 6361 ggttggaaca gctggagaca tggtgagagg taccacccca accctcgtcctcgccatgcg 6421 ctgtgatttg taagttgcag tgccctgcat atagcaagag atactgttctctatttgtct 6481 ctgctcccca gaatagagcc ctgctccctg cctgactgca gctctattctgcctcctcag 6541 cctcaccacg cagggaagcc cagaagtccc agtctccttc agggaaaggaatgaattaac 6601 ccacaatctg gttttgcttc ttttttttaa tcacccagaa atatatatatatgtattttt 6661 tttttactgc aacgaataca atgacaagaa aggaagggaa ggaaggaaggaagagaaaat 6721 tacctattac ctagcttatt aaacaaaaat ggaatcatat tgtccatactattttgaaat 6781 ccatggggtt ttttttaagc ttaacagtat tttatatata tatatatatatatatatata 6841 tatatatata tatatatata tatatttttt tttttttttt ttttttttttttttgagacg 6901 gagtctctct ctgttccctg gctggcggag cggagtcggc acgatctcagctcactgcaa 6961 cttccaactc ccacggttca agccaattct cctgtctcag cctcccgagcctgggattac 7021 caggcacaca ccagcctggc tagttttttt gattttttag tagagacgatgtttctccat 7081 gttggccagg ctggtctcaa actcctgact tcaggtgatc cacccaacttgggctcccaa 7141 agtgctggga ttacaggcgt gacgaccatg cccggccaac agtatattatatttatccat 7201 gttatttctt atgtccacac aacagtcccc tatatggtgg taacataatttaattaatga 7261 actcctattt tcagctattt aggttatttt caatttcttg ttaccttttgccaggaaacg 7321 tatattttat ggtaattata ttgtgttgta gaaaaatcac tagtctagtccaacttgctt 7381 gaaaaatagc tactttttaa ctattttctc atttaaaaat ttattataatttagtctttt 7441 agaaatatac caggccaggc atggcgtctc atgcctgtta tcctagtactttggaaggct 7501 gaggacggag gatcacttca gtcttggggt ttgagaccag cccgggaaacataacaagac 7561 cccatctcta caaaaaaaaa aaattgtttt taattaggca tgtccgacacagtggctcac 7621 acatgtggcc agcactgtgg gaaggccaag gtgggtggat cacttgagggtcaggagttc 7681 aagaccagcc tggccaatgt ggtgaaaccc catctctact aaaaatacaaaaatttgcca 7741 ggtgtggtgg cgcatgcctg tattcccagc tactcaggag gctaaggcaggaaatcactt 7801 gaactcggag gcagaggttg cagtgagctg tgacaatgcc actgtactccagcctgggtg 7861 acagagcgag ctccgtctca aaaaaaaaaa aaaaagatta ggcatggtggcacacgcctg 7921 tagaccctag ctactcagga ggctgaggtg ggaggattgc ttgagcccaggtgttggagg 7981 ctgcagtgag ccatgattat accactgtag tccagcctgg acaacagaacgagaccctgt 8041 ctctaaaagt atatatgtac acataccata atacccagct actgaggaggctgaggcaga 8101 aagagtgctt gagtccagga gtttgatgtc agcctgagca atatagcaagaccctcacct 8161 cttaaaaaaa tttaaagtag attaaaaaaa taccacaatt gctcaggtagattaaaaaaa 8221 taccacaatt gctcaggtag attattgaaa aacaggcata tagtacttatggtacaggac 8281 cagcatgcat gcatgcatgc attgattgat tgattgattg attgattgagacagggtctc 8341 tctctgtctc ccaggctgga gtgcctggcc ttaagtgatc tgcccacctttgcttcccaa 8401 agtgctgaga ttacaggtgt gagccaccat gtcagctggc gaggctttttaaaagatagt 8461 tccaagtgtt acagctcttt taggatttgt ctagcaggct ttcaggtttttgccagaaac 8521 cacccccacc cccaccaaaa aaaaaaaaaa aaaaaagata tgtacaagttcccagatagt 8581 gttcccaact gaatctattt ctcatgtgta gtgtatggtt gttttcctgtcaccacattg 8641 ctgattatta ttatttttaa ttatagagac agtaaagtac agtagttaaaaatgtgagtt 8701 ggggctgggt gcagtggctc acacctgtaa tcccagcact ttgggaggccaaggtgggcg 8761 gatcacctga ggtcaggagt tcaagaccag cttggccaac atggcaaaaccccgtctcga 8821 ctaaaaatat atatatataa gttagccggg cgtggtggca acattacctgtaatcccagc 8881 tactcgggag gccaacaggc aggagaatct cttgaatcca ggaggtggaggttgcagtga 8941 gccagatcac accattgcac tccagcctgg atgacaagag agtgagactgtctaaaaaaa 9001 aaaaacaaag tgtgagttgt acaatgagac tgcctgggat cacatacaagcttcatccct 9061 tactagttgt attgacccta aagcaagtca ctaacctttc tgtgccctccagttttatca 9121 tctgtaatgt ggggaaaata atagtacctg cctcagaggg ttgttttgaggattaaatgc 9181 attaatatgt ggaaagggct taatataagt tgtacatagc atatgaaaactgttatgtta 9241 aatctattag cagttttata tgtgaaaata gctttgattt tcatttcttggattatgaat 9301 catgttgaat aatcctttat atgcttcctg gattcttttt ttttcttccccccagtcagt 9361 ttctgactct tctcatattt atagagagat cttggaacct ggatgggggaatccaggaaa 9421 ctcatggatt ccttcttcct gaattttatc acccaggttc acagctggagcaaagctgtt 9481 gtttcacctg aggcagctgc tgaaggagct gaagggactg agttgcctggttagctatca 9541 ggatgaccct ctgaccaaag gggtggacct acgcaacgcc caggtcacagagttgctaca 9601 gcgtctgctc cacaggtcta gaggccaggc aggaaccctg ggggaaagaaggaacaaggg 9661 aagccattct tacacatact gagctatata ttctctccac acctctctctcctcgagcct 9721 ttgtggtaga aacccagccc tgcatgcccc aaactcccca tcgacccctcatcctcaaga 9781 ctggcagcaa gttcaccgtc cgaacaaggt tggcattcca gaactcattcccacttcctt 9841 tttccaaccc tgccactgtg tattttctgg ctttacagct actgcccactcttggctttt 9901 tcagtctttc ctgaatctcc ctacctcgtt gataccccat cgtcctctttttcaaacacc 9961 tagcctatac aaaagccgac tccgaccaca tttccctata ccccttgacttccccaggct 10021 gctggtgaga ctccaggaag gcaatgagtc actgactgtg gaagtctccattgacaggta 10081 aattggagca ggtgaagggt ggccaggaca cgggctgctg gggtggaggagatactcact 10141 cttcacaaca gggccctagg gctatatcct tcctccttcc aatcctacctcacagaaatt 10201 ataattcatt tcttttgttg aacacttact ttgtgacatg cagcatgtcagctactcatt 10261 taattgtcac accaacccca tgaataaact attaccagtg cactgtacaaacaaagatac 10321 aggcttagag agactgatta catctcttct caaggccaca tagctagtgagctcaagtcg 10381 ggtttgaacc gaggtctgtc tgatcccaaa gacgaaactc ctaacttccatactcttttg 10441 cccaatgatt ttttttaaat ttatttcttt tcaggaatcc tcctcaattacaagggtagg 10501 tgcttgacaa ggacactgca aacatctgta cagtgtatga cctgcagaaccgggggattt 10561 gggaaatgga caaagggaga tggcgagatc tgaaatggaa gtggaacttcagtttttttt 10621 ttttctgctg agtttttaca ataattccat tccttgtctc catgtatcttcctcctggaa 10681 cagcttccgg aagttcaaca ttctgacttc aaaccagaaa actttgacccccgagaaggg 10741 gcagagtcag ggtttgattt gggactttgg ttacctggta agaatagtttgtgacctatg 10801 cttttattac tatttttatt ttttcgagac ggagtctcac tctgtcccccaggctggagt 10861 gcagtggtgc catcttggct cacaggaacc tccgccctcc ccggttcaagcaattcttct 10921 gtctcagcct cctgagtacg tagagctata ggcagcacac caccatgcccggctaatttt 10981 tgtattttta gtagagatag ggtttcacca tattggtcgg gctggtctcgaactcctgac 11041 ctcaggtgat ccgacccgcc tcagcctccc aaagtgctgg gatcacaggcatgagccacc 11101 atagctggcc tgcttttagt ccaaaggaac aggggttggg ggaagttcccagggcttgag 11161 aggtcttgaa gccaaacagg ggttccaggg agactagggt gcccactctggcattttctc 11221 tccttccctt caattcacag actctggtgg agcaacgttc aggtggttcaggaaagggca 11281 gcaataaggt gagatctgga cagaggactc gaggcagggg gagcttgccaaagagccttc 11341 tgatgactat gtctttgcct gtcccagagg ggccactagg tgtgacagaggaactgcaca 11401 tcatcagctt cacggtcaaa tatacctacc agggtctgaa gcaggagctgaaagtgagtg 11461 aaaatggagg gcaaggagag agaaagcagc tttggaagaa ggcataagaaggggataaac 11521 agaagcctct tggggagggt tagcactcct ttcctctaac aaatacctgcagctagaaac 11581 atcacatccc tctctgtgac tcctgtcttc tccccacaca cggacaccctccctgtggtg 11641 attatttcca acatgaacca gctctcaatt gcctgggctt cagttctctggttcaatttg 11701 ctcagcccaa accttcaggt aggggagtgg ggccgacagg tcccggcgcgagagcagggg 11761 tgtggaagct tggtgtgata ggttgcttct gagccagcct acactgctcccacccctgca 11821 gaaccagcag ttcttctcca acccccccaa ggccccctgg agcttgctgggccctgctct 11881 cagttggcag ttctcctcct atgttggccg aggcctcaac tcagaccagctgagcatgct 11941 gagaaacaag ctgttcggta cagatttcct tttctctcag cctttccccagccttagtct 12001 tttctgtccc tctgtcctat ctatcccagg acccctggct tccctcacatatctgtggct 12061 atctgtccca cagggcagaa ctgtaggact gaggatccat tattgtcctgggctgacttc 12121 actaaggtaa ctccctgaat cctgtggagc tgctggatct agccccacattccaaatact 12181 ggccttccca cgtgccctcc ttccctacac cagaggcaac tcctcagcttttgctacctt 12241 tccattcctc cagcgagaga gccctcctgg caagttacca ttctggacatggctggacaa 12301 aattctggag ttggtacatg accacctgaa ggatctctgg aatgatgggtaaggccttgg 12361 tcacccttcc ctcatgggct tgtgcttccg ggcttgagag tggagtctctgcaccctcac 12421 gtggcaagca gggagagaga gcaaagcacg gtgcaggcca cgtctcctcacatttgttaa 12481 gaataataag gccgggtgtg gtggctcaca cctgtaatcc cagcactttgggaggccgag 12541 gcgggcggat catgaggtca ggagatcgag accatcctgg ctaacacggtgaaaccccgt 12601 ctctactcta aaaatacaaa aaattagccg ggcgtggagg cagacaccctgtagtcccag 12661 ctactcagga ggctgaggca ggaaaatggc gtgaacctgg gagatggagcttgcagtgag 12721 ccgagattgc gtcactgccc tccagccttg gggtgacgta gcaagactccgtctcaaaaa 12781 aaaaaaaaaa aaacaaccaa taatagccat aaacagtgtt tttgtgaagcactcctacat 12841 tccagagctt gatgggtgct cttcattaat tctctcatct catccttacaaccatgctga 12901 gtggtgggtt ttgccagctt catttcatgt gaggaaactg agtttcagagaagttaaaga 12961 acttacccaa gggacacagt tgatattcaa atccaggcct atgtgactccaagcccatgc 13021 tctttccacc acactgccta ccaacttgtg tagcatttgg cttttaaaagtgctattcat 13081 gaccaggcac gatggctcac gccttgtaat cccagcattt tgggaggccgaggtgggtgg 13141 atcacctgag gtcaggagtt tgagaccagc ctggccaaca tggcgaaaccccatctctat 13201 taaaaataca aaaattagcc gggtgtggtg gtgggcgcct gtaatcccagctactcagga 13261 ggctgaggag gagaatcgct tgaatttagg agagaaggtt acagtgagccaagatcgtgc 13321 cattgcactc cagcctgggt gacagagcaa gactctgtct caaaacaaaaccaaaaaaaa 13381 gtgctatttg tggccaggcg tggttgctca tgcctgtaat cctagcatttttggggaggc 13441 tgaggagtac agatcacttg agcccaggag ttcaaaacta ccctgggccacgtggtgaaa 13501 ccccaaaccc cgtctctacg aaaaatacaa aagttagcca ggatgggtggtgtgcacctg 13561 tggtcccagc tactctggag gctgagaggt ggggaagatt gcttgagcccgggaggtcga 13621 ggtggcagtg agctgtgatc atgccactat tctccagcct gggtgacagaatacaccctg 13681 tctccctgtc tcccagaaaa aaaaaaaagt gctgttcatc tgtgtgatctcactgaatct 13741 tcgtacttca aaccctcgga aggtggctat tgtcagcaaa gtgaagtgacttgtaaaaga 13801 taaaaaaaag ctaagtggca gggcttggtc caaagcctgg attccaaacctgggctgttt 13861 ctccatacaa ggggagcagg gaggcagggg cctggggggg cagggtgttgggcggtgtca 13921 cacgtgacac actgtgctcc agacgcatca tgggctttgt gagtcggagccaggagcgcc 13981 ggctgctgaa gaagaccatg tctggcacct ttctactgcg cttcagtgaatcgtcagaag 14041 ggggcattac ctgctcctgg gtggagcacc aggatgatgg tagctgctctgccctgccat 14101 tcccacagcc tctcctttct gcctggctct cctctggccc ctctgcctgccttgcttcgc 14161 tggctctgaa ctgaatgctc agtggtttgg gactgggcag ccagagagtcagagagctcc 14221 aaggcccggc ctcttccctc aagcccgcct gttcctgcat tcactctccagacaaggtgc 14281 tcatctactc tgtgcaaccg tacacgaagg aggtgctgca gtcactcccgctgactgaaa 14341 tcatccgcca ttaccagttg ctcactgagg agaatatacc tgaaaacccactgcgcttcc 14401 tctatccccg aatcccccgg gatgaagctt ttgggtgcta ctaccaggagaaaggtggga 14461 atcgttgaca tacttcattg ctagattgca gagatctacc agacatccatagatcccact 14521 ccttccttta aagcatggga aaactgatat ctagaggaat taagggattcgtccatggga 14581 tactgctggt tactatgggg atgagactgc caggaccatc tgcactaggggaaaacctca 14641 ggctatatgt ctggcccact gatcttctct gcttcttgta tatgttcctcacagttaatc 14701 tccaggaacg gaggaaatac ctgaaacaca ggctcattgt ggtctctaatagagtgagat 14761 atgaactgtt cattcatcct ccctaatcct tattggctct gcttcagtgaatcgtcaaaa 14821 gggggcatta ccttctcctg ggtggagcac caggatgatg gtcagctgctctgccctgcc 14881 attcccacag cctctccttt ctgccttctc ctaagctgcc cctattccagtctccccagc 14941 cttccctccc tcctagcccc actctagttt tttctggttc tagtctctcctatctcatat 15001 ttttctgctg ccatccttag gttgtctcca caggggtttc tggataataatgatcataat 15061 cactggtgtt aaggggtacc tacttgatgc aagcatggag ctttttttttttccagacag 15121 ggttttgttc tgtcgcccag gctggagtgc agtggtgtga tcctggctcactgcagcctc 15181 gacctcctga gctcaagcaa tacaggcatg catcaccaaa ctcagctaattttttttgta 15241 ttttttgtag agatggggtc ttaccatgtt gacgcatcag gctgttctgaactcctggac 15301 tcaagcaatc cacccacctt ggcctcccaa aagtcaggga ttacaggcgtgcgaccacac 15361 cccgcatata tatatttttt tttttttttt tttttttttt tttttgagacagggtctctg 15421 ttatccaggc tggagttgca gtggataata tgactacgag ccttgacctaggggttgaag 15481 caatgctcct gcctcagcca ccaagtgctg agactacagg cacacgccaatctacactca 15541 atcacactca gctaattttt taaatttttt gtagggatgg ggtatcactgtgtttgccca 15601 ggctggtctt gaactcctgg cctcaagcag tctcctgcct tggcctcccaaattgccggg 15661 attgtaggaa tgagccatgg cacttggctg ggggatagaa tttttttttttttttttttt 15721 tttttttttt ttgagacagt ctcactctca ttgcccgggc tggagtgcagtggtgcaatt 15781 tcagctcact gcaacctctg cctcccaggc tcaagcaatt ctcctgcctcagcctataga 15841 gtagctggga ttacaggcga gcgccaccca tgcctggtta atttttgttttttttttgag 15901 acagagtctc gccctgttgc ccaggctgga gtgcagtggc acgatctcagctcactgcaa 15961 cctctgcctc ccaggctcaa gcaattctcc tgcctcagcc tcctgagtactgggactaca 16021 agcgcgcaca accaccacac ctggtaattt ttgtattttt agtagagacagggttttacc 16081 atattggcca ggctggtctc aaactcctga cctcatgatc cgacccaccttggcctccca 16141 aagtgcaggg attacaggcg tgagcctctg cacccggcct aacttttgtatttttagtag 16201 aaacagggtt tcaccatgtt ggccaggctg gtcatgagct cctggcctcaagtgatctgc 16261 ccgcctcagc ctcccaaagt gcttggatta caggtgtgag ccacctggcctgagagttta 16321 ttatgcgcca ggcactaggc aaatggtttg catttatttt ctcattttattgaatctaca 16381 aaatagtcct gtgaagtaaa cactgttact gttttcagct aaggaactggatttagagta 16441 gtcaagtttt gtacctaagg tacgtggcta atgatacagg tctgttagattccgtagccc 16501 tgattttaac caccctactg cctctcaaga attactaggt attgttctcatttatagatg 16561 ataaatctga ggctcagaaa agttaggcca cttgcctaag gtcccccagccaggattcaa 16621 actccaggag gcctgattcc aaacccatgc tctttagccc tccgccctactgccttctta 16681 gactagcttc tgcttattct accattcctg atttcatttg aaccactgagccctgcccct 16741 ttgtctgtct ttgggtatcc aggcaggtgg atgaactgca acaaccgctggagcttaagc 16801 cagagccaga gctggagtca ttagagctgg aactagggct ggtgccagagccagagctca 16861 gcctggactt agagccactg ctgaaggcag ggctggatct ggggccagagctagagtctg 16921 tgctggagtc cactctggag cctgtgatag agcccacact atgcatggtatcacaaacag 16981 tgccagagcc agaccaagga cctgtatcac agccagtgcc agagccagatttgccctgtg 17041 atctgagaca tttgaacact gagccaatgg aaagtaagtg atgagatggagtggcacaca 17101 ttccctttcc tacctcttct ccctctccca ttacagaaaa agctgaactccaagctcctc 17161 attggagaga ggtccatctg tgattccttt ttttaggaat tacacatgccttcccccacc 17221 tccctgctct ttcatcccac aagttcccac tcaggctctt cccaggcctttcctgccatc 17281 ctccctccct tgggctgctg ggttgggaac tcctaactaa gatcggggcctcacttttct 17341 ctctggatta cctagtcttc agaaactgtg taaagattga agaaatcatgccgaatggtg 17401 acccactgtt ggctggccag aacaccgtgg atgaggttta cgtctcccgccccagccact 17461 tctacactga tggacccttg atgccttctg acttctagga accacatttcctctgttctt 17521 ttcatatctc tttgcccttc ctactcctca tagcatgata ttgttctccaaggatgggaa 17581 tcaggcatgt gtcccttcca agctgtgtta actgttcaaa ctcaggcctgtgtgactcca 17641 ttggggtgag aggtgaaagc ataacatggg tacagagggg acaacaatgaatcagaacag 17701 atgctgagcc ataggtctaa ataggatcct ggaggctgcc tgctgtgctgggaggtatag 17761 gggtcctggg ggcaggccag ggcagttgac aggtacttgg agggctcagggcagtggctt 17821 ctttccagta tggaaggatt tcaacatttt aatagttggt taggctaaactggtgcatac 17881 tggcattggc cttggtgggg agcacagaca caggatagga ctccatttctttcttccatt 17941 ccttcatgtc taggataact tgctttcttc tttcctttac tcctggctcaagccctgaat 18001 ttcttctttt cctgcagggg ttgagagctt tctgccttag cctaccatgtgaaactctac 18061 cctgaagaaa gggatggata ggaagtagac ctctttttct taccagtctcctcccctact 18121 ctgcccccta agctggctgt acctgttcct cccccataaa atgatcctgccaatctaatg 18181 tgagtgtgaa gtttgcacac tagtttatgc tacctagtct ccactttctcaatgcttagg 18241 agacagatca ctcctggagg ctggggatgg taggattgct ggggatttttttttttttaa 18301 agagggtctc actctgttgc ccaggctaga gtgcaatggt gcaatcacagctcactgcag 18361 cctcaacctc ctgggttcaa gcaatcctcc tacctcagcc tcctgggtagctagcaccat 18421 ggcatcgcca ccatgcccta tttttttttt ttaaagacag ggtcttgctatattgcccag 18481 gctggtcttg aactgggctc aagtgatcct cacgccttgc ctcccaaagtgctgggatta 18541 taggcatgag ccactgtgct tggccaggat tttttttttt ttttttttgagatggagttt 18601 ctctcttgtt gtccaggctg gagtgcaatg gtgtgatccg gggaattc

Protein variants can include amino acid sequence modifications. Forexample, amino acid sequence modifications fall into one or more ofthree classes: substitutional, insertional or deletional variants.Insertions can include amino and/or carboxyl terminal fusions as well asintrasequence insertions of single or multiple amino acid residues.Insertions ordinarily will be smaller insertions than those of amino orcarboxyl terminal fusions, for example, on the order of one to fourresidues. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. These variants ordinarilyare prepared by site-specific mutagenesis of nucleotides in the DNAencoding the protein, thereby producing DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture.

Nucleic acid sequences comprising a gene, such as a Jak1, Jak2, Jak3,Stat 1, and/or Stat 2 gene, that encodes a polypeptide can besynthesized, in whole or in part, using chemical methods known in theart. Alternatively, a polypeptide, such as a Jak1, Jak2, Jak3, Stat 1,and/or Stat 2, can be produced using chemical methods to synthesize itsamino acid sequence, such as by direct peptide synthesis usingsolid-phase techniques. Protein synthesis can either be performed usingmanual techniques or by automation. Automated synthesis can be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Optionally, fragments of Jak1, Jak2, Jak3, Stat 1, and/or Stat 2polypeptides can be separately synthesized and combined using chemicalmethods to produce a full-length molecule.

As used herein, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 “molecule” can bea nucleic acid which encodes a polypeptide that exhibits Jak1, Jak2,Jak3, Stat 1, and/or Stat 2 activity, or a polypeptide or peptidomimeticthat exhibits Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity. Forexample, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can include thehuman Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein, or a variant thereof,such as a fragment thereof, that exhibits Jak1, Jak2, Jak3, Stat 1,and/or Stat 2 activity. Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activitycan encompass signaling events by way of type I cytokine receptors(e.g., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) that use the common gammachain (γc). For example, Jak1, Jak2, Jak3, Stat 1, and/or Stat 2activity can be a signal transduced in response to its activation viatyrosine phosphorylation by interleukin receptors.

The nucleic acid can be any type of nucleic acid, including genomic DNA,complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well asany form of corresponding RNA. For example, a Jak1, Jak2, Jak3, Stat 1,or Stat 2 molecule can comprise a recombinant nucleic acid encodinghuman Jak1, Jak2, Jak3, Stat 1, or Stat 2 protein. In one embodiment, aJak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can comprise anon-naturally occurring nucleic acid created artificially (such as byassembling, cutting, ligating or amplifying sequences). A Jak1, Jak2,Jak3, Stat 1, or Stat 2 molecule can be double-stranded. A Jak1, Jak2,Jak3, Stat 1, or Stat 2 molecule can be single-stranded. The Jak1, Jak2,Jak3, Stat 1, and/or Stat 2 molecules of the invention can be obtainedfrom various sources and can be produced according to various techniquesknown in the art. For example, a nucleic acid that is a Jak1, Jak2,Jak3, Stat 1, or Stat 2 molecule can be obtained by screening DNAlibraries, or by amplification from a natural source. The Jak1, Jak2,Jak3, Stat 1, and/or Stat 2 molecules can be produced via recombinantDNA technology and such recombinant nucleic acids can be prepared byconventional techniques, including chemical synthesis, geneticengineering, enzymatic techniques, or a combination thereof.Non-limiting examples of a Jak1, Jak2, Jak3, Stat 1, or Stat 2 moleculethat is a nucleic acid. Another example of a Jak1, Jak2, Jak3, Stat 1,or Stat 2 molecule is a fragment of a nucleic acid, wherein the fragmentexhibits Jak1, Jak2, Jak3, Stat 1, and/or Stat 2 activity. A Jak1, Jak2,Jak3, Stat 1, or Stat 2 molecule of this invention also encompassesvariants of the human nucleic acid encoding the Jak1, Jak2, Jak3, Stat1, or Stat 2 protein, or variants of the human Jak1, Jak2, Jak3, Stat 1,or Stat 2 proteins that exhibit Jak1, Jak2, Jak3, Stat 1, and/or Stat 2activity. A Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can alsoinclude a fragment of the human Jak1, Jak2, Jak3, Stat 1, or Stat 2nucleic acid which encodes a polypeptide that exhibits Jak1, Jak2, Jak3,Stat 1, and/or Stat 2 activity. A Jak1, Jak2, Jak3, Stat 1, or Stat 2molecule can encompass a fragment of the human Jak1, Jak2, Jak3, Stat 1,or Stat 2 protein that exhibits Jak1, Jak2, Jak3, Stat 1, and/or Stat 2activity.

A Jak1, Jak2, Jak3, Stat 1, or Stat 2 molecule can also encompass Jak1,Jak2, Jak3, Stat 1, or Stat 2 ortholog genes, which are genes conservedamong different biological species such as humans, dogs, cats, mice, andrats, that encode proteins (for example, homologs (including splicevariants), mutants, and derivatives) having biologically equivalentfunctions as the human-derived protein (such as a Jak3 protein). Jak1,Jak2, Jak3, Stat 1, or Stat 2 orthologs include any mammalian orthologof Jak1, Jak2, Jak3, Stat 1, or Stat 2 inclusive of the ortholog inhumans and other primates, experimental mammals (such as mice, rats,hamsters and guinea pigs), mammals of commercial significance (such ashorses, cows, camels, pigs and sheep), and also companion mammals (suchas domestic animals, e.g., rabbits, ferrets, dogs, and cats).

The Jak1, Jak2, Jak3, Stat 1, and Stat 2 variants can comprise, forinstance, naturally-occurring variants due to allelic variations betweenindividuals (e.g., polymorphisms), mutated alleles related to alopeciaareata, or alternative splicing forms. In one embodiment, a Jak1, Jak2,Jak3, Stat 1, or Stat 2 molecule is a nucleic acid variant of thenucleic acid having the sequence shown in herein, wherein the varianthas a nucleotide sequence identity to a corresponding sequence disclosedherein of about 65%, about 75%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,or about 99%. In one embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2molecule encompasses any portion of about 8 consecutive nucleotides of acorresponding sequence disclosed herein. In one embodiment, the fragmentcan comprise about 15 nucleotides, about 20 nucleotides, or about 30nucleotides of a corresponding sequence disclosed herein. Fragmentsinclude all possible nucleotide lengths between about 8 and 100nucleotides, for example, lengths between about 15 and 100, or betweenabout 20 and 100.

The invention further provides for nucleic acids that are complementaryto a nucleic acid encoding a Jak1, Jak2, Jak3, Stat 1, or Stat 2protein. Such complementary nucleic acids can comprise nucleic acidsequences, which hybridize to a nucleic acid sequence encoding a Jak1,Jak2, Jak3, Stat 1, or Stat 2 protein under stringent hybridizationconditions. Non-limiting examples of stringent hybridization conditionsinclude temperatures above 30° C., above 35° C., in excess of 42° C.,and/or salinity of less than about 500 mM, or less than 200 mM.Hybridization conditions can be adjusted by the skilled artisan viamodifying the temperature, salinity and/or the concentration of otherreagents such as SDS or SSC.

In one embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 moleculecomprises a protein or polypeptide encoded by a Jak1, Jak2, Jak3, Stat1, or Stat 2 nucleic acid sequence, such as the corresponding sequencedisclosed herein. In another embodiment, the polypeptide can bemodified, such as by glycosylations and/or acetylations and/or chemicalreaction or coupling, and can contain one or several non-natural orsynthetic amino acids. An example of a Jak1, Jak2, Jak3, Stat 1, or Stat2 molecule is the polypeptide having the amino acid sequence disclosedherein. In another embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2molecule can be a fragment of a Jak1, Jak2, Jak3, Stat 1, or Stat 2protein. For example, the Jak1, Jak2, Jak3, Stat 1, or Stat 2 moleculecan encompass any portion of about 8 consecutive amino acids of thecorresponding sequence disclosed herein. The fragment can comprise about10 amino acids, a least about 20 amino acids, about 30 amino acids,about 40 amino acids, a least about 50 amino acids, about 60 aminoacids, or about 75 amino acids of the corresponding sequence disclosedherein. Fragments include all possible amino acid lengths between about8 and 100 about amino acids, for example, lengths between about 10 and100 amino acids, between about 15 and 100 amino acids, between about 20and 100 amino acids, between about 35 and 100 amino acids, between about40 and 100 amino acids, between about 50 and 100 amino acids, betweenabout 70 and 100 amino acids, between about 75 and 100 amino acids, orbetween about 80 and 100 amino acids.

In certain embodiments, the Jak1, Jak2, Jak3, Stat 1, or Stat 2 moleculeincludes variants of the human Jak1, Jak2, Jak3, Stat 1, or Stat 2protein (comprising the corresponding sequence disclosed herein). Suchvariants can include those having at least from about 46% to about 50%identity to the corresponding sequence disclosed herein, or having atleast from about 50.1% to about 55% identity to the correspondingsequence disclosed herein, or having at least from about 55.1% to about60% identity to SEQ ID NO: 1, or having from about 60.1% to about 65%identity to the corresponding sequence disclosed herein, or having fromabout 65.1% to about 70% identity to the corresponding sequencedisclosed herein, or having at least from about 70.1% to about 75%identity to the corresponding sequence disclosed herein, or having atleast from about 75.1% to about 80% identity to the correspondingsequence disclosed herein, or having at least from about 80.1% to about85% identity to the corresponding sequence disclosed herein, or havingat least from about 85.1% to about 90% identity to the correspondingsequence disclosed herein, or having at least from about 90.1% to about95% identity to the corresponding sequence disclosed herein, or havingat least from about 95.1% to about 97% identity to the correspondingsequence disclosed herein, or having at least from about 97.1% to about99% identity to the corresponding sequence disclosed herein.

Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example M13 primermutagenesis and PCR mutagenesis. Amino acid substitutions can be singleresidues, but can occur at a number of different locations at once. Inone non-limiting embodiment, insertions can be on the order of aboutfrom 1 to about 10 amino acid residues, while deletions can range fromabout 1 to about 30 residues. Deletions or insertions can be made inadjacent pairs (for example, a deletion of about 2 residues or insertionof about 2 residues). Substitutions, deletions, insertions, or anycombination thereof can be combined to arrive at a final construct. Themutations cannot place the sequence out of reading frame and should notcreate complementary regions that can produce secondary mRNA structure.Substitutional variants are those in which at least one residue has beenremoved and a different residue inserted in its place.

Substantial changes in function or immunological identity are made byselecting residues that differ more significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site or (c)the bulk of the side chain. The substitutions that can produce thegreatest changes in the protein properties will be those in which (a) ahydrophilic residue, e.g. seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine, in this case, (e) byincreasing the number of sites for sulfation and/or glycosylation.

There can be minor variations in the amino acid sequences of thecorresponding sequence disclosed herein for each of Jak1, Jak2, Jak3,Stat 1, and/or Stat 2. The variations in the amino acid sequence can bewhen the sequence maintains about 30%, about 40%, about 50%, about 60%,about 70%, about 75%, about 80%, about 90%, about 95%, or about 99%identity to the corresponding sequence disclosed herein. For example,conservative amino acid replacements can be utilized. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains, wherein the interchangeability ofresidues have similar side chains.

Genetically encoded amino acids are generally divided into families: (1)acidic amino acids are aspartate, glutamate; (2) basic amino acids arelysine, arginine, histidine; (3) non-polar amino acids are alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan, and (4) uncharged polar amino acids are glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic aminoacids include arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) a group of amino acids havingaliphatic-hydroxyl side chains, such as serine and threonine; (ii) agroup of amino acids having amide-containing side chains, such asasparagine and glutamine; (iii) a group of amino acids having aliphaticside chains such as glycine, alanine, valine, leucine, and isoleucine;(iv) a group of amino acids having aromatic side chains, such asphenylalanine, tyrosine, and tryptophan; and (v) a group of amino acidshaving sulfur-containing side chains, such as cysteine and methionine.Useful conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine valine, glutamic-aspartic, and asparagine-glutamine.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Substitutional or deletional mutagenesiscan be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) orO-glycosylation (Ser or Thr). Deletions of cysteine or other labileresidues also can be desirable. Deletions or substitutions of potentialproteolysis sites, e.g. Arg, is accomplished for example by deleting oneof the basic residues or substituting one by glutaminyl or histidylresidues.

In another embodiment, the Jak1, Jak2, Jak3, Stat 1, or Stat 2 moleculeencompasses a peptidomimetic which exhibits Jak1, Jak2, Jak3, Stat 1, orStat 2 activity. A peptidomimetic is a small protein-like chain designedto mimic a peptide that can arise from modification of an existingpeptide in order to protect that molecule from enzyme degradation andincrease its stability, and/or alter the molecule's properties (e.g.,modifications that change the molecule's stability or biologicalactivity). These modifications involve changes to the peptide thatcannot occur naturally (such as altered backbones and the incorporationof non-natural amino acids). Drug-like compounds can be developed fromexisting peptides. A peptidomimetic can be a peptide, partial peptide,or non-peptide molecule that mimics the tertiary binding structure oractivity of a selected native peptide or protein functional domain(e.g., binding motif or active site). These peptide mimetics includerecombinantly or chemically modified peptides.

In one embodiment, a Jak1, Jak2, Jak3, Stat 1, or Stat 2 moleculecomprising the corresponding sequence disclosed herein, variants ofsuch, or fragments thereof, can be modified to produce peptide mimeticsby replacement of one or more naturally occurring side chains of the 20genetically encoded amino acids (or D amino acids) with other sidechains. This can occur, for instance, with groups such as alkyl, loweralkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl,amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lowerester derivatives thereof, and with 4, 5-, 6-, to 7-memberedheterocyclics. For example, proline analogs can be made in which thering size of the proline residue is changed from 5 members to 4, 6, or 7members. Cyclic groups can be saturated or unsaturated, and ifunsaturated, can be aromatic or non-aromatic. Heterocyclic groups cancontain one or more nitrogen, oxygen, and/or sulphur heteroatoms.Examples of such groups include the furazanyl, ifuryl, imidazolidinylimidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g.1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,thienyl, thiomorpholinyl (e.g. thiomorpholino), and triazolyl. Theseheterocyclic groups can be substituted or unsubstituted. Where a groupis substituted, the substituent can be alkyl, alkoxy, halogen, oxygen,or substituted or unsubstituted phenyl. Peptidomimetics can also haveamino acid residues that have been chemically modified byphosphorylation, sulfonation, biotinylation, or the addition or removalof other moieties. For example, peptidomimetics can be designed anddirected to amino acid sequences encoded by a Jak1, Jak2, Jak3, Stat 1,or Stat 2 molecule comprising the corresponding sequence disclosedherein.

A variety of techniques are available for constructing peptide mimeticswith the same or similar desired biological activity as thecorresponding native but with more favorable activity than the peptidewith respect to solubility, stability, and/or susceptibility tohydrolysis or proteolysis (see, e.g., Morgan & Gainor, Ann. Rep. Med.Chem. 24, 243-252, 1989). Certain peptidomimetic compounds are basedupon the amino acid sequence of the peptides of the invention.Peptidomimetic compounds can be synthetic compounds having athree-dimensional structure (i.e. a peptide motif) based upon thethree-dimensional structure of a selected peptide. The peptide motifprovides the peptidomimetic compound with the desired biologicalactivity, wherein the binding activity of the mimetic compound is notsubstantially reduced, and is often the same as or greater than theactivity of the native peptide on which the mimetic is modeled.Peptidomimetic compounds can have additional characteristics thatenhance their therapeutic application, such as increased cellpermeability, greater affinity and/or avidity and prolonged biologicalhalf-life. Peptidomimetic design strategies are readily available in theart (see, e.g., Ripka & Rich (1998) Curr. Op. Chem. Biol. 2:441-452;Hruby et al. (1997) Curr. Op. Chem. Biol. 1:114-119; Hruby & Balse,(2000) Curr. Med. Chem. 9:945-970).

Cell Transfection

A eukaryotic expression vector can be used to transfect cells in orderto produce proteins encoded by nucleotide sequences of the vector.Mammalian cells (such as isolated cells from the hair bulb; for exampledermal sheath cells and dermal papilla cells) can contain an expressionvector (for example, one that contains a gene encoding a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein or polypeptide) via introducing theexpression vector into an appropriate host cell via methods known in theart.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedpolypeptide encoded by a gene, such as a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 gene, in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of thepolypeptide also can be used to facilitate correct insertion, foldingand/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and W138), are available fromthe American Type Culture Collection (ATCC; 10801 University Boulevard,Manassas, Va. 20110-2209) and can be chosen to ensure the correctmodification and processing of the foreign protein.

An exogenous nucleic acid can be introduced into a cell via a variety oftechniques known in the art, such as lipofection, microinjection,calcium phosphate or calcium chloride precipitation,DEAE-dextran-mediated transfection, or electroporation. Electroporationis carried out at approximate voltage and capacitance to result in entryof the DNA construct(s) into cells of interest (such as cells of the endbulb of a hair follicle, for example dermal papilla cells or dermalsheath cells). Other transfection methods also include modified calciumphosphate precipitation, polybrene precipitation, liposome fusion, andreceptor-mediated gene delivery.

Cells that will be genetically engineered can be primary and secondarycells obtained from various tissues, and include cell types which can bemaintained and propagated in culture. Non-limiting examples of primaryand secondary cells include epithelial cells (for example, dermalpapilla cells, hair follicle cells, inner root sheath cells, outer rootsheath cells, sebaceous gland cells, epidermal matrix cells), neuralcells, endothelial cells, glial cells, fibroblasts, muscle cells (suchas myoblasts) keratinocytes, formed elements of the blood (e.g.,lymphocytes, bone marrow cells), and precursors of these somatic celltypes.

Vertebrate tissue can be obtained by methods known to one skilled in theart, such a punch biopsy or other surgical methods of obtaining a tissuesource of the primary cell type of interest. In one embodiment, a punchbiopsy or removal can be used to obtain a source of keratinocytes,fibroblasts, endothelial cells, or mesenchymal cells (for example, hairfollicle cells or dermal papilla cells). In another embodiment, removalof a hair follicle can be used to obtain a source of fibroblasts,keratinocytes, endothelial cells, or mesenchymal cells (for example,hair follicle cells or dermal papilla cells). A mixture of primary cellscan be obtained from the tissue, using methods readily practiced in theart, such as explanting or enzymatic digestion (for examples usingenzymes such as pronase, trypsin, collagenase, elastase dispase, andchymotrypsin). Biopsy methods have also been described in United StatesPatent Application Publication 2004/0057937 and PCT applicationpublication WO 2001/32840, and are hereby incorporated by reference.

Primary cells can be acquired from the individual to whom thegenetically engineered primary or secondary cells are administered.However, primary cells can also be obtained from a donor, other than therecipient, of the same species. The cells can also be obtained fromanother species (for example, rabbit, cat, mouse, rat, sheep, goat, dog,horse, cow, bird, or pig). Primary cells can also include cells from anisolated vertebrate tissue source grown attached to a tissue culturesubstrate (for example, flask or dish) or grown in a suspension; cellspresent in an explant derived from tissue; both of the aforementionedcell types plated for the first time; and cell culture suspensionsderived from these plated cells. Secondary cells can be plated primarycells that are removed from the culture substrate and replated, orpassaged, in addition to cells from the subsequent passages. Secondarycells can be passaged one or more times. These primary or secondarycells can contain expression vectors having a gene that encodes aprotein of interest (for example, a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2protein or polypeptide).

Cell Culturing

Various culturing parameters can be used with respect to the host cellbeing cultured. Appropriate culture conditions for mammalian cells arewell known in the art (Cleveland W L, et al., J Immunol Methods, 1983,56(2): 221-234) or can be determined by the skilled artisan (see, forexample, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D.and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cellculturing conditions can vary according to the type of host cellselected. Commercially available medium can be utilized. Non-limitingexamples of medium include, for example, Minimal Essential Medium (MEM,Sigma, St. Louis, Mo.); Dulbecco's Modified Eagles Medium (DMEM, Sigma);Ham's F10 Medium (Sigma); HyClone cell culture medium (HyClone, Logan,Utah); RPMI-1640 Medium (Sigma); and chemically-defined (CD) media,which are formulated for various cell types, e.g., CD-CHO Medium(Invitrogen, Carlsbad, Calif.).

The cell culture media can be supplemented as necessary withsupplementary components or ingredients, including optional components,in appropriate concentrations or amounts, as necessary or desired. Cellculture medium solutions provide at least one component from one or moreof the following categories: (1) an energy source, usually in the formof a carbohydrate such as glucose; (2) all essential amino acids, andusually the basic set of twenty amino acids plus cysteine; (3) vitaminsand/or other organic compounds required at low concentrations; (4) freefatty acids or lipids, for example linoleic acid; and (5) traceelements, where trace elements are defined as inorganic compounds ornaturally occurring elements that can be required at very lowconcentrations, usually in the micromolar range.

The medium also can be supplemented electively with one or morecomponents from any of the following categories: (1) salts, for example,magnesium, calcium, and phosphate; (2) hormones and other growth factorssuch as, serum, insulin, transferrin, and epidermal growth factor; (3)protein and tissue hydrolysates, for example peptone or peptone mixtureswhich can be obtained from purified gelatin, plant material, or animalbyproducts; (4) nucleosides and bases such as, adenosine, thymidine, andhypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such asgentamycin or ampicillin; (7) cell protective agents, for examplepluronic polyol; and (8) galactose. In one embodiment, soluble factorscan be added to the culturing medium.

The mammalian cell culture that can be used with the present inventionis prepared in a medium suitable for the type of cell being cultured. Inone embodiment, the cell culture medium can be any one of thosepreviously discussed (for example, MEM) that is supplemented with serumfrom a mammalian source (for example, fetal bovine serum (FBS)). Inanother embodiment, the medium can be a conditioned medium to sustainthe growth of epithelial cells or cells obtained from the hair bulb of ahair follicle (such as dermal papilla cells or dermal sheath cells). Forexample, epithelial cells can be cultured according to Barnes and Matherin Animal Cell Culture Methods (Academic Press, 1998), which is herebyincorporated by reference in its entirety. In a further embodiment,epithelial cells or hair follicle cells can be transfected with DNAvectors containing genes that encode a polypeptide or protein ofinterest (for example, a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein orpolypeptide). In other embodiments of the invention, cells are grown ina suspension culture (for example, a three-dimensional culture such as ahanging drop culture) in the presence of an effective amount of enzyme,wherein the enzyme substrate is an extracellular matrix molecule in thesuspension culture. For example, the enzyme can be a hyaluronidase.Epithelial cells or hair follicle cells can be cultivated according tomethods practiced in the art, for example, as those described in PCTapplication publication WO 2004/044188 and in U.S. Patent ApplicationPublication No. 2005/0272150, or as described by Harris in Handbook inPractical Animal Cell Biology: Epithelial Cell Culture (Cambridge Univ.Press, Great Britain; 1996; see Chapter 8), which are herebyincorporated by reference.

A suspension culture is a type of culture wherein cells, or aggregatesof cells (such as aggregates of DP cells), multiply while suspended inliquid medium. A suspension culture comprising mammalian cells can beused for the maintenance of cell types that do not adhere or to enablecells to manifest specific cellular characteristics that are not seen inthe adherent form. Some types of suspension cultures can includethree-dimensional cultures or a hanging drop culture. A hanging-dropculture is a culture in which the material to be cultivated isinoculated into a drop of fluid attached to a flat surface (such as acoverglass, glass slide, Petri dish, flask, and the like), and can beinverted over a hollow surface. Cells in a hanging drop can aggregatetoward the hanging center of a drop as a result of gravity. However,according to the methods of the invention, cells cultured in thepresence of a protein that degrades the extracellular matrix (such ascollagenase, chondroitinase, hyaluronidase, and the like) will becomemore compact and aggregated within the hanging drop culture, fordegradation of the ECM will allow cells to become closer in proximity toone another since less of the ECM will be present. See alsoInternational PCT Publication No. WO2007/100870, which is incorporatedby reference.

Cells obtained from the hair bulb of a hair follicle (such as dermalpapilla cells or dermal sheath cells) can be cultured as a single,homogenous population (for example, comprising DP cells) in a hangingdrop culture so as to generate an aggregate of DP cells. Cells can alsobe cultured as a heterogeneous population (for example, comprising DPand DS cells) in a hanging drop culture so as to generate a chimericaggregate of DP and DS cells. Epithelial cells can be cultured as amonolayer to confluency as practiced in the art. Such culturing methodscan be carried out essentially according to methods described in Chapter8 of the Handbook in Practical Animal Cell Biology: Epithelial CellCulture (Cambridge Univ. Press, Great Britain; 1996); Underhill C B, JInvest Dermatol, 1993, 101(6):820-6); in Armstrong and Armstrong, (1990)J Cell Biol 110:1439-55; or in Animal Cell Culture Methods (AcademicPress, 1998), which are all hereby incorporated by reference in theirentireties.

Three-dimensional cultures can be formed from agar (such as Gey's Agar),hydrogels (such as matrigel, agarose, and the like; Lee et al., (2004)Biomaterials 25: 2461-2466) or polymers that are cross-linked. Thesepolymers can comprise natural polymers and their derivatives, syntheticpolymers and their derivatives, or a combination thereof. Naturalpolymers can be anionic polymers, cationic polymers, amphipathicpolymers, or neutral polymers. Non-limiting examples of anionic polymerscan include hyaluronic acid, alginic acid (alginate), carageenan,chondroitin sulfate, dextran sulfate, and pectin. Some examples ofcationic polymers, include but are not limited to, chitosan orpolylysine. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A.S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NYAcad Sci 944: 62-73). Examples of amphipathic polymers can include, butare not limited to collagen, gelatin, fibrin, and carboxymethyl chitin.Non-limiting examples of neutral polymers can include dextran, agarose,or pullulan. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A.S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NYAcad Sci 944: 62-73).

Cells suitable for culturing according to methods of the invention canharbor introduced expression vectors, such as plasmids. The expressionvector constructs can be introduced via transformation, microinjection,transfection, lipofection, electroporation, or infection. The expressionvectors can contain coding sequences, or portions thereof, encoding theproteins for expression and production. Expression vectors containingsequences encoding the produced proteins and polypeptides, as well asthe appropriate transcriptional and translational control elements, canbe generated using methods well known to and practiced by those skilledin the art. These methods include synthetic techniques, in vitrorecombinant DNA techniques, and in vivo genetic recombination which aredescribed in J. Sambrook et al., 2001, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubelet al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

Obtaining and Purifying Polypeptides

A polypeptide molecule encoded by a gene, such as a Jak 1, Jak 2, Jak3,Stat 1, or Stat 2 gene, or a variant thereof, can be obtained bypurification from human cells expressing a protein or polypeptideencoded by a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 gene via in vitro orin vivo expression of a nucleic acid sequence encoding a Jak 1, Jak 2,Jak3, Stat 1, or Stat 2 protein or polypeptide; or by direct chemicalsynthesis.

Detecting Polypeptide Expression.

Host cells which contain a nucleic acid encoding a Jak 1, Jak 2, Jak3,Stat 1, or Stat 2 protein or polypeptide, and which subsequently expressa protein encoded by a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 gene, canbe identified by various procedures known to those of skill in the art.These procedures include, but are not limited to, DNA-DNA or DNA-RNAhybridizations and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip-based technologies for the detectionand/or quantification of nucleic acid or protein. For example, thepresence of a nucleic acid encoding a Jak 1, Jak 2, Jak3, Stat 1, orStat 2 protein or polypeptide can be detected by DNA-DNA or DNA-RNAhybridization or amplification using probes or fragments of nucleicacids encoding a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 protein orpolypeptide. In one embodiment, a fragment of a nucleic acid of a Jak 1,Jak 2, Jak3, Stat 1, or Stat 2 gene can encompass any portion of atleast about 8 consecutive nucleotides of the corresponding sequencedisclosed herein. In another embodiment, the fragment can comprise atleast about 10 consecutive nucleotides, at least about 15 consecutivenucleotides, at least about 20 consecutive nucleotides, or at leastabout 30 consecutive nucleotides of the corresponding sequence disclosedherein. Fragments can include all possible nucleotide lengths betweenabout 8 and about 100 nucleotides, for example, lengths between about 15and about 100 nucleotides, or between about 20 and about 100nucleotides. Nucleic acid amplification-based assays involve the use ofoligonucleotides selected from sequences encoding a polypeptide encodedby a Jak 1, Jak 2, Jak3, Stat 1, or Stat 2 gene to detect transformantswhich contain a nucleic acid encoding a Jak 1, Jak 2, Jak3, Stat 1, orStat 2 protein or polypeptide.

Protocols for detecting and measuring the expression of a polypeptideencoded by a gene, such as a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene,using either polyclonal or monoclonal antibodies specific for thepolypeptide are well established. Non-limiting examples includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay using monoclonal antibodies reactive to two non-interferingepitopes on a polypeptide encoded by a gene, such as a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 gene, can be used, or a competitive binding assaycan be employed.

Labeling and conjugation techniques are known by those skilled in theart and can be used in various nucleic acid and amino acid assays.Methods for producing labeled hybridization or PCR probes for detectingsequences related to nucleic acid sequences encoding a protein, such asJak 1, Jak 2, Jak3, Stat 1 or Stat 2, include, but are not limited to,oligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, nucleic acid sequencesencoding a polypeptide encoded by a gene, such as a Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 gene, can be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and can be used to synthesize RNA probes in vitro by additionof labeled nucleotides and an appropriate RNA polymerase such as T7, T3,or SP6. These procedures can be conducted using a variety ofcommercially available kits (Amersham Pharmacia Biotech, Promega, and USBiochemical). Suitable reporter molecules or labels which can be usedfor ease of detection include radionuclides, enzymes, and fluorescent,chemiluminescent, or chromogenic agents, as well as substrates,cofactors, inhibitors, and/or magnetic particles.

Expression and Purification of Polypeptides.

Host cells transformed with a nucleic acid sequence encoding apolypeptide, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat 2, can becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The polypeptide produced by a transformedcell can be secreted or contained intracellularly depending on thesequence and/or the vector used. Expression vectors containing a nucleicacid sequence encoding a polypeptide, such as Jak 1, Jak 2, Jak3, Stat 1or Stat 2, can be designed to contain signal sequences which directsecretion of soluble polypeptide molecules encoded by a gene, such as aJak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, or a variant thereof, througha prokaryotic or eukaryotic cell membrane or which direct the membraneinsertion of membrane-bound a polypeptide molecule encoded by a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 gene or a variant thereof.

Other constructions can also be used to join a gene sequence encoding aJak 1, Jak 2, Jak3, Stat 1 or Stat 2 polypeptide to a nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). Including cleavablelinker sequences (i.e., those specific for Factor Xa or enterokinase(Invitrogen, San Diego, Calif.)) between the purification domain and apolypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene alsocan be used to facilitate purification. One such expression vectorprovides for expression of a fusion protein containing a polypeptideencoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene and 6 histidine(SEQ ID NO: 111) residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification byimmobilized metal ion affinity chromatography, while the enterokinasecleavage site provides a means for purifying the polypeptide encoded bya Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene.

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 polypeptide can be purified fromany human or non-human cell which expresses the polypeptide, includingthose which have been transfected with expression constructs thatexpress a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein. A purified Jak1, Jak 2, Jak3, Stat 1 or Stat 2 protein can be separated from othercompounds which normally associate with a protein encoded by a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 gene in the cell, such as certainproteins, carbohydrates, or lipids, using methods practiced in the art.Non-limiting methods include size exclusion chromatography, ammoniumsulfate fractionation, ion exchange chromatography, affinitychromatography, and preparative gel electrophoresis.

Chemical Synthesis.

Nucleic acid sequences comprising a gene, such as a Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 gene, that encodes a polypeptide can be synthesized, inwhole or in part, using chemical methods known in the art.Alternatively, a polypeptide, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat2, can be produced using chemical methods to synthesize its amino acidsequence, such as by direct peptide synthesis using solid-phasetechniques. Protein synthesis can either be performed using manualtechniques or by automation. Automated synthesis can be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Optionally, fragments of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2polypeptides can be separately synthesized and combined using chemicalmethods to produce a full-length molecule. In one embodiment, a fragmentof a nucleic acid sequence that comprises a Jak 1, Jak 2, Jak3, Stat 1or Stat 2 gene can encompass any portion of at least about 8 consecutivenucleotides of the corresponding sequence disclosed herein. In oneembodiment, the fragment can comprise at least about 10 nucleotides, atleast about 15 nucleotides, at least about 20 nucleotides, or at leastabout 30 nucleotides of the corresponding sequence disclosed herein.Fragments include all possible nucleotide lengths between about 8 andabout 100 nucleotides, for example, lengths between about 15 and about100 nucleotides, or between about 20 and about 100 nucleotides.

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 fragment can be a fragment of aprotein, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat 2. For example, theJak 1, Jak 2, Jak3, Stat 1 or Stat 2 fragment can encompass any portionof at least about 8 consecutive amino acids of the correspondingsequence disclosed herein. The fragment can comprise at least about 10consecutive amino acids, at least about 20 consecutive amino acids, atleast about 30 consecutive amino acids, at least about 40 consecutiveamino acids, a least about 50 consecutive amino acids, at least about 60consecutive amino acids, at least about 70 consecutive amino acids, orat least about 75 consecutive amino acids of the corresponding sequencedisclosed herein. Fragments include all possible amino acid lengthsbetween about 8 and 100 about amino acids, for example, lengths betweenabout 10 and about 100 amino acids, between about 15 and about 100 aminoacids, between about 20 and about 100 amino acids, between about 35 andabout 100 amino acids, between about 40 and about 100 amino acids,between about 50 and about 100 amino acids, between about 70 and about100 amino acids, between about 75 and about 100 amino acids, or betweenabout 80 and about 100 amino acids.

A synthetic peptide can be substantially purified via high performanceliquid chromatography (HPLC). The composition of a synthetic polypeptideof Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 can be confirmed by amino acidanalysis or sequencing. Additionally, any portion of an amino acidsequence comprising a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 gene can be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins to produce a variantpolypeptide or a fusion protein.

Identifying Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 Modulating Compounds

The invention provides methods for identifying compounds which can beused for controlling and/or regulating hair growth (for example, hairdensity) or hair pigmentation in a subject. Since the invention hasprovided the identification of the genes listed herein as genesassociated with a hair loss disorder, the invention also providesmethods for identifying compounds that modulate the expression oractivity of a gene and/or protein of Jak 1, Jak 2, Jak3, Stat 1 or Stat2. In addition, the invention provides methods for identifying compoundswhich can be used for the treatment of a hair loss disorder. Theinvention also provides methods for identifying compounds which can beused for the treatment of hypotrichosis (for example, hereditaryhypotrichosis simplex (HHS)). Non-limiting examples of hair lossdisorders include: androgenetic alopecia, Alopecia areata, telogeneffluvium, alopecia areata, alopecia totalis, and alopecia universalis.The methods can comprise the identification of test compounds or agents(e.g., peptides (such as antibodies or fragments thereof), smallmolecules, nucleic acids (such as siRNA or antisense RNA), or otheragents) that can bind to a polypeptide molecule encoded by a Jak 1, Jak2, Jak3, Stat 1 or Stat 2 gene and/or have a stimulatory or inhibitoryeffect on the biological activity of a protein encoded by a Jak 1, Jak2, Jak3, Stat 1 or Stat 2 gene or its expression, and subsequentlydetermining whether these compounds can regulate hair growth in asubject or can have an effect on symptoms associated with the hair lossdisorders in an in vivo assay (i.e., examining an increase or reductionin hair growth).

As used herein, a “Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulatingcompound” refers to a compound that interacts with a Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 gene or a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteinor polypeptide and modulates its activity and/or its expression. Thecompound can either increase the activity or expression of a proteinencoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. Conversely, thecompound can decrease the activity or expression of a protein encoded bya Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. The compound can be a Jak1, Jak 2, Jak3, Stat 1 or Stat 2 agonist or a Jak 1, Jak 2, Jak3, Stat 1or Stat 2 antagonist (e.g., a Jak1 inhibitor, a Jak2 inhibitor, a Stat1inhibitor, or a Stat2 inhibitor). Some non-limiting examples of Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds include peptides(such as peptide fragments comprising a polypeptide encoded by a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 gene, or antibodies or fragments thereof),small molecules, and nucleic acids (such as siRNA or antisense RNAspecific for a nucleic acid comprising a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 gene). Agonists of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteincan be molecules which, when bound to a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 protein, increase or prolong the activity of the Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein. Jak 1, Jak 2, Jak3, Stat 1 or Stat 2agonists include, but are not limited to, proteins, nucleic acids, smallmolecules, or any other molecule which activates a Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 protein. Antagonists of a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 protein can be molecules which, when bound to a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein decrease the amount or the duration ofthe activity of the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein.Antagonists include proteins, nucleic acids, antibodies, smallmolecules, or any other molecule which decrease the activity of a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 protein.

The term “modulate,” as it appears herein, refers to a change in theactivity or expression of a gene or protein of Jak 1, Jak 2, Jak3, Stat1 or Stat 2. For example, modulation can cause an increase or a decreasein protein activity, binding characteristics, or any other biological,functional, or immunological properties of a Jak 1, Jak 2, Jak3, Stat 1or Stat 2 protein.

In one embodiment, a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulatingcompound can be a peptide fragment of a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 protein that binds to the protein. For example, the Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 polypeptide can encompass any portion of at leastabout 8 consecutive amino acids of the corresponding sequence disclosedherein. The fragment can comprise at least about 10 consecutive aminoacids, at least about 20 consecutive amino acids, at least about 30consecutive amino acids, at least about 40 consecutive amino acids, atleast about 50 consecutive amino acids, at least about 60 consecutiveamino acids, or at least about 75 consecutive amino acids of thecorresponding sequence disclosed herein. Fragments include all possibleamino acid lengths between and including about 8 and about 100 aminoacids, for example, lengths between about 10 and about 100 amino acids,between about 15 and about 100 amino acids, between about 20 and about100 amino acids, between about 35 and about 100 amino acids, betweenabout 40 and about 100 amino acids, between about 50 and about 100 aminoacids, between about 70 and about 100 amino acids, between about 75 andabout 100 amino acids, or between about 80 and about 100 amino acids.These peptide fragments can be obtained commercially or synthesized vialiquid phase or solid phase synthesis methods (Atherton et al., (1989)Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford,England). The Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 peptide fragments canbe isolated from a natural source, genetically engineered, or chemicallyprepared. These methods are well known in the art.

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can be aprotein, such as an antibody (monoclonal, polyclonal, humanized,chimeric, or fully human), or a binding fragment thereof, directedagainst a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2gene. An antibody fragment can be a form of an antibody other than thefull-length form and includes portions or components that exist withinfull-length antibodies, in addition to antibody fragments that have beenengineered. Antibody fragments can include, but are not limited to,single chain Fv (scFv), diabodies, Fv, and (Fab′)₂, triabodies, Fc, Fab,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, tetrabodies,bifunctional hybrid antibodies, framework regions, constant regions, andthe like (see, Maynard et al., (2000) Ann. Rev. Biomed. Eng. 2:339-76;Hudson (1998) Curr. Opin. Biotechnol. 9:395-402). Antibodies can beobtained commercially, custom generated, or synthesized against anantigen of interest according to methods established in the art (Janewayet al., (2001) Immunobiology, 5th ed., Garland Publishing). In oneembodiment, an antibody or binding fragment thereof is directed againstSEQ ID NO: 1, 3, 5 or 7. Antibodies can be obtained commercially, customgenerated, or synthesized against an antigen of interest according tomethods established in the art (Janeway et al., (2001) Immunobiology,5th ed., Garland Publishing). For example, antibodies directed to Jak1,Jak2, Stat1, or Stat2 can be obtained commercially from Abcam, SantaCruz Biotechnology, Abnova Corp., BD Biosciences, Antigenix AmericaInc., etc. Human antibodies directed to either Jak1, Jak2, Stat1, orStat2 (such as monoclonal, humanized, or chimeric antibodies) can beuseful antibody therapeutics for use in humans.

Inhibition of RNA encoding a polypeptide encoded by a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 gene can effectively modulate the expression of aJak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene from which the RNA istranscribed. Inhibitors are selected from the group comprising: siRNA;interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs;ribozymes; and antisense nucleic acids, which can be RNA, DNA, or anartificial nucleic acid.

Antisense oligonucleotides, including antisense DNA, RNA, and DNA/RNAmolecules, act to directly block the translation of mRNA by binding totargeted mRNA and preventing protein translation. For example, antisenseoligonucleotides of at least about 15 bases and complementary to uniqueregions of the DNA sequence encoding a polypeptide encoded by a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 gene can be synthesized, e.g., byconventional phosphodiester techniques (Dallas et al., (2006) Med. Sci.Monit. 12(4):RA67-74; Kalota et al., (2006) Handb. Exp. Pharmacol.173:173-96; Lutzelburger et al., (2006) Handb. Exp. Pharmacol.173:243-59). Antisense nucleotide sequences include, but are not limitedto: morpholinos, 2′-O-methyl polynucleotides, DNA, RNA and the like.

siRNA comprises a double stranded structure containing from about 15 toabout 50 base pairs, for example from about 21 to about 25 base pairs,and having a nucleotide sequence identical or nearly identical to anexpressed target gene or RNA within the cell. The siRNA comprise a senseRNA strand and a complementary antisense RNA strand annealed together bystandard Watson-Crick base-pairing interactions. The sense strandcomprises a nucleic acid sequence which is substantially identical to anucleic acid sequence contained within the target miRNA molecule.“Substantially identical” to a target sequence contained within thetarget mRNA refers to a nucleic acid sequence that differs from thetarget sequence by about 3% or less. The sense and antisense strands ofthe siRNA can comprise two complementary, single-stranded RNA molecules,or can comprise a single molecule in which two complementary portionsare base-paired and are covalently linked by a single-stranded “hairpin”area. See also, McMnaus and Sharp (2002) Nat Rev Genetics, 3:737-47, andSen and Blau (2006) FASEB J., 20:1293-99, the entire disclosures ofwhich are herein incorporated by reference.

The siRNA can be altered RNA that differs from naturally-occurring RNAby the addition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA, or modifications that make the siRNA resistantto nuclease digestion, or the substitution of one or more nucleotides inthe siRNA with deoxyribonucleotides. One or both strands of the siRNAcan also comprise a 3′ overhang. As used herein, a 3′ overhang refers toat least one unpaired nucleotide extending from the 3′-end of a duplexedRNA strand. For example, the siRNA can comprise at least one 3′ overhangof from 1 to about 6 nucleotides (which includes ribonucleotides ordeoxyribonucleotides) in length, or from 1 to about 5 nucleotides inlength, or from 1 to about 4 nucleotides in length, or from about 2 toabout 4 nucleotides in length. For example, each strand of the siRNA cancomprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid(“uu”).

siRNA can be produced chemically or biologically, or can be expressedfrom a recombinant plasmid or viral vector (for example, see U.S. Pat.Nos. 7,294,504 and 7,422,896, the entire disclosures of which are hereinincorporated by reference). Exemplary methods for producing and testingdsRNA or siRNA molecules are described in U.S. Patent ApplicationPublication No. 2002/0173478 to Gewirtz, U.S. Patent ApplicationPublication No. 2007/0072204 to Hannon et al., and in U.S. PatentApplication Publication No. 2004/0018176 to Reich et al., the entiredisclosures of which are herein incorporated by reference.

In one embodiment, an siRNA directed to a human nucleic acid sequencecomprising a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can be generatedagainst any one of SEQ ID NOS: 2, 4, 6 or 8. In another embodiment, ansiRNA directed to a human nucleic acid sequence comprising a Jak1 genecan comprise any one of the sequences listed in Table 1. In anotherembodiment, an siRNA directed to a human nucleic acid sequencecomprising a Stat1 gene can comprise any one of the sequences listed inTable 2. In another embodiment, an siRNA directed to a human nucleicacid sequence comprising a Jak3 gene can comprise any one of thesequences listed in Table 3.

In another embodiment, the siRNA directed to Jak1 is listed in Table 1.

TABLE 1 siRNA SEQUENCES for Jak1 (SEQ ID NOS: 9-32, (SEQ ID NOS:respectively, in order 33-58, respectively, in of appearance) order ofappearance) CCACATAGCTGATCTGAAA TCTGAAGAGAAGAAGATAA CGGGAAGCCTTAAGGAATAGCAAGAATGCATTGAACGA CTGAAGAGAAGAAGATAAA AAGAAAGATTGATGGACTTACAAGAAGGATGAGGAGAA AAAATAAACTGAAGCGGAA CCAAGAAGACTGAGGTGAAATGAGGAAATGCTGGGAAT CAGAATACGCCATCAATAA AAAGAAAGATTGATGGACTGCACAGAAGACGGAGGAAA GGACTTAGCCCTCAAATTT GCGATATATTCCAGAAACAACACTGGACAGCTGAATAA TGAGCTACTTGGAGGATAA CTGAATAAATGCAGTATCTGAGTGAACACCAAGTGAAA AAGAATGCATTGAACGAAT CAAGAAGGATGAGGAGAAACCAGTAACTTAGTGACACA CGGGAAGAGTGGAACAATT TTAACAAGCAGGACAACAAGCATTAACAAGCAGGACAA GGGAGCAGGTGGCTGTTAA TTTGGAAAGTAGAGAAGAAAAATCGAGATCTTAAGGAA GGAAAGTAGAGAAGAAAAT GGAAATGGTATTAAGCTCAGGAACCACGCTCTGGGAAA CCAGAATGTTTAATGCAAT TGATTGAGAAAGAGAGATTGGACAAGCTTTCAGAACCT ACATTACGGTGCTGAAATA ACACATAATGACAACCAAAGGGAAGAGTGGAACAATTT GCTTGGAGGTAGCTGGGTA AGAATATCATGGTGGAAGACAGCAGAGGAACTGTGCAT GGAAATGTGTGTACTAAAA CCAGAAACATTGAATAAGTATGAGAACATTGTGAAGTA CTGAAATATTTGAGACTTC CACAGAAGACGGAGGAAATGTACAGAATACGCCATCAA GCTGGTGGCTACTAAGAAA GCACCGACTTTGACAACATGAGAAGAAGATAAAAGTGA GGAAATTCAAAGTTGCCAA

In another embodiment, the siRNA directed to Stat1 is listed in Table 2.

TABLE 2 siRNA SEQUENCES for Stat1 (SEQ ID NOS: 59-81, (SEQ ID NOS:82-108, respectively, in order respectively, in of appearance) order ofappearance) AGAAAGAGCTTGACAGTAA TGAAATTGCAAGAGCTGAA AGGAATTGGAACAGAAATAAGAAATACACCTACGAACA CAGCATAACATAAGGAAAA CCCTAAAGGAACTGGATATTCAGAAATGTGAAGGACAA GGAGGAATTGGAACASAAA GGCAAAGAGTGATCAGAAACCTAAAGGAACTGGATATA GCACAGTGATGTTAGACAA AGCGTAATCTTCAGGATAAAGTCATGGCTGCTGAGAAT TCTGAAGGAAGAAAGGAAA GTATAGAGCATGAAATCAAGGAAGATTTACAAGATGAA GGTTATGTGTATAGAGCAT TGGCAAAGAGTGATCAGAAGAAAGGAAGCAGTTCACAA AGAGAAAGGAAGTAGTTCA GATGTGAATGAGAGAAATATAATAGAGTTGCTGAATGT ACAGAGAACACGAGACCAA TGGAGGAATTGGAACAGAAAAGATGTGAATGAGAGAAA AGTTGAGACTGTTGGTGAA AGGACAAGGTTATGTGTATGATAAAGATGTGAATGAGA CCTGATTAATGATGAACTA CATCGTTACTGAAGAGCTTACACAAAAGTGATGAACAT TGAAGTATCTGATCCAAA GCAATTGAAAGAACAGAAAGCACAAGGTGGCAGGATGT CGAGAGCTGTCTAGGTTAA TGTCACAGCTGGATGATCAGAACATGACCCTATCACAA GAAAGAGCTTGACAGTAAA AGACAAACAGAAAGAGCTTTCAAGAGCCTGGAAGATTT GGACAAGGTTATGTGTATA TGGAAGATTTACAAGATGATGACAATAAGAGAAAGGAA TGAGACTGTTGGTGAAATT GGAAGTAGTTCACAAAATATTGCAAGAGCTGAATTATA GGATTTAGGAAGTTCAACA GGAACTTGATGGCCC AAAGGAACTGGATATATCAAGA

In another embodiment, the siRNA directed to Jak3 is listed in Table 3.

TABLE 3 siRNA SEQUENCES for Jak3 siRNA sequences TCAACTATCTGGAGGACAA(SEQ ID NO: 112) AGACAGAGGTGCTGCTGAA (SEQ ID NO: 113)GGTCCTTCACCAAGATTTA (SEQ ID NO: 114) CCTGGATCCTGCTAAGAAA (SEQ ID NO:115) CGATCTTCGAGGAGAGACA (SEQ ID NO: 116) GGACAGACAACCAGATTTT(SEQ ID NO:117) GGAAGCTGCAGGTGGTCAA (SEQ ID NO: 118) CCCAATACCAGCTGAGTCA (SEQ IDNO: 119)

RNA polymerase III transcribed DNAs contain promoters, such as the U6promoter. These DNAs can be transcribed to produce small hairpin RNAs inthe cell that can function as siRNA or linear RNAs that can function asantisense RNA. The Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulatingcompound can contain ribonucleotides, deoxyribonucleotides, syntheticnucleotides, or any suitable combination such that the target RNA and/orgene is inhibited. In addition, these forms of nucleic acid can besingle, double, triple, or quadruple stranded. (see for example Bass(2001) Nature, 411, 428 429; Elbashir et al., (2001) Nature, 411, 494498; and PCT Publication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO00/01846, WO 01/29058, WO 99/07409, WO 00/44914).

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can be asmall molecule that binds to a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2protein and disrupts its function, or conversely, enhances its function.Small molecules are a diverse group of synthetic and natural substancesgenerally having low molecular weights. They can be isolated fromnatural sources (for example, plants, fungi, microbes and the like), areobtained commercially and/or available as libraries or collections, orsynthesized. Candidate small molecules that modulate a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein can be identified via in silico screeningor high-through-put (HTP) screening of combinatorial libraries (e.g.,see Potyrailo et al., (2011)ACS Comb Sci. 13(6):579-633; Mensch et al.,(2009) J Pharm Sci. 98(12):4429-68; Schnur (2008) Curr Opin Drug DiscovDevel. 11(3):375-80; and Jhoti (2007) Ernst Schering Found Symp Proc.(3):169-85, each of which are hereby incorporated by reference in theirentireties). Most conventional pharmaceuticals, such as aspirin,penicillin, and many chemotherapeutics, are small molecules, can beobtained commercially, can be chemically synthesized, or can be obtainedfrom random or combinatorial libraries as described below (Werner etal., (2006) Brief Funct. Genomic Proteomic 5(1):32-6).

Non-limiting examples of Jak1/Jak2 inhibitors include: AG490(Caceres-Cortes, Anticancer Agents Med Chem. 2008 October; 8(7):717-22);CYT387 (Pardanani et al., Leukemia. 2009 August; 23(8):1441-5; Monaghanet al., Leukemia. 2011 Jul. 26. doi: 10.1038/leu.2011.175. [Epub aheadof print]); SB1518 (William et al., J Med Chem. 2011 Jul. 14;54(13):4638-58; Hart et al., Leukemia. 2011 Jun. 21. doi:10.1038/leu.2011.148. [Epub ahead of print]); LY3009104 (INCB28050)(Incyte and Lilly); TG101348 (Wernig et al., Cancer Cell. 2008 April;13(4):311-20; Pardanani et al., J Clin Oncol. 2011 Mar. 1;29(7):789-96); and BMS-911543 (Purandare et al., Leukemia. 2011 Oct. 21.doi: 10.1038/leu.2011.292. [Epub ahead of print]), each of thereferences of which are incorporated by reference in their entireties.

JAK1/2 inhibitors in clinical development include a) INCB018424, topicaland oral; 5 nM activity (Incyte); b) CEP-701 (Cephalon); and c)TG101348.

JAK3 inhibitors are currently in clinical trials in humans for thetreatment of acute kidney transplant rejection and rheumatoid arthritis.Non-limiting examples of Jak3 inhibitors include: Janex 1 (WHI-P131)(Changelian et al., (2008) Blood, 111(4):2155-7); Uckun ey al., (1999)Clin Cancer Res. 5(10):2954-62; Uckun et al., (2010)Arzneimittelforschung. 60(4):218-25), PF-956980 (Sigma Product # PZ0151(St. Louis, Mo., sigmaaldrich.com/catalog/product/sigma/pz0151?lang=en&region=US)); Changelian et al., (2008) Blood, 111(4):2155-7),WHI-P154 (Calbiochem Product #420104-5MG (San Diego, Calif.,emdbiosciences.com)); Changelian et al., (2008) Blood, 111(4):2155-7),VX-509 (oral from Vertex Pharmaceuticals, Cambridge Mass.; Fleischmannet al. (2011) Arthritis Rheum, 63:LB3; Fleischmann et al., (2012) CurrOpin Rheumatol. February 18, PMID: 22357358), JAK3 Inhibitor IV(ZM-39923) Calbiochem Product #420121-10MG (San Diego, Calif.,emdbiosciences.com, WO1998022103), NSC114792 (Kim et al., (2010)MolCancer. 2010, 9:36), tofacitinib (CP690550) (Changelian et al., (2008)Blood, 111(4):2155-7; Vijayakrishnan et al. (2011) Trends Pharmacol Sci.32(1):25-34; Fleischmann et al., (2012) Curr Opin Rheumatol. February18, PMID: 22357358), and R348 (topical and oral from RigelPharmaceuticals, San Francisco Calif.; Deuse et al., (2008)Transplantation. 85(6):885-92; Vijayakrishnan et al. (2011) TrendsPharmacol Sci. 32(1):25-34), each of the references of which areincorporated by reference in their entireties.

Structures of JAK3 inhibitors useful for the invention include a) Janex1, oral and topical; b) PF-956980, i.v. infusion; c) WHI-P154; d)ZM-39923; e) NSC114792; f) tofacitinib (CP690550), oral.

for example

Non-limiting examples of JAK inhibitors (for example, Type I and Type IIJak Inhibitors) are discussed in O'Shea and Plenge (Immunity, 2012 Apr.20; 36(4):542-50), LaFave and Levine (Trends Pharmacol Sci. 2012November; 33(11):574-82), Kontzias et al, (Curr Opin Pharmacol. 2012August; 12(4):464-70), Norman (Expert Opin Ther Pat. 2012 October;22(10):1233-49), and Wilson (Expert Opin Ther Pat. 2010 May;20(5):609-23), each of which are hereby incorporated by reference intheir entireties.

Non-limiting examples of Stat inhibitors include: WP-1034 (Faderl etal., Anticancer Res. 2005 May-June; 25(3B):1841-50), fludarabine(Fludara, Berlex, Calif.), epigallocatechin-3-gallate (EGCG), andHyperforin. Other compounds directed to Jak/Stat signaling are describedin Ivanenkov et al., Mini Rev Med Chem. 2011 January; 11(1):55-78, thecontents of which are incorporated by reference in its entirety.

Knowledge of the primary sequence of a molecule of interest, such as apolypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, andthe similarity of that sequence with proteins of known function, canprovide information as to the inhibitors or antagonists of the proteinof interest in addition to agonists. Identification and screening ofagonists and antagonists is further facilitated by determiningstructural features of the protein, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry, and othertechniques for structure determination. These techniques provide for therational design or identification of agonists and antagonists.

Test compounds, such as Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulatingcompounds, can be screened from large libraries of synthetic or naturalcompounds (see Wang et al., (2007) Curr Med Chem, 14(2):133-55; Mannhold(2006) Curr Top Med Chem, 6 (10):1031-47; and Hensen (2006) Curr MedChem 13(4):361-76). Numerous means are currently used for random anddirected synthesis of saccharide, peptide, and nucleic acid basedcompounds. Synthetic compound libraries are commercially available fromMaybridge Chemical Co. (Trevillet, Cornwall, UK), AMRI (Albany, N.Y.),ChemBridge (San Diego, Calif.), and MicroSource (Gaylordsville, Conn.).A rare chemical library is available from Aldrich (Milwaukee, Wis.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available from e.g. PanLaboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readilyproducible. Additionally, natural and synthetically produced librariesand compounds are readily modified through conventional chemical,physical, and biochemical means (Blondelle et al., (1996) Tib Tech14:60).

Methods for preparing libraries of molecules are well known in the artand many libraries are commercially available. Libraries of interest inthe invention include peptide libraries, randomized oligonucleotidelibraries, synthetic organic combinatorial libraries, and the like.Degenerate peptide libraries can be readily prepared in solution, inimmobilized form as bacterial flagella peptide display libraries or asphage display libraries. Peptide ligands can be selected fromcombinatorial libraries of peptides containing at least one amino acid.Libraries can be synthesized of peptoids and non-peptide syntheticmoieties. Such libraries can further be synthesized which containnon-peptide synthetic moieties, which are less subject to enzymaticdegradation compared to their naturally-occurring counterparts. Forexample, libraries can also include, but are not limited to,peptide-on-plasmid libraries, synthetic small molecule libraries,aptamer libraries, in vitro translation-based libraries, polysomelibraries, synthetic peptide libraries, neurotransmitter libraries, andchemical libraries.

Examples of chemically synthesized libraries are described in Fodor etal., (1991) Science 251:767-773; Houghten et al., (1991) Nature354:84-86; Lam et al., (1991) Nature 354:82-84; Medynski, (1994)BioTechnology 12:709-710; Gallop et al., (1994)J. Medicinal Chemistry37(9):1233-1251; Ohlmeyer et al., (1993) Proc. Natl. Acad. Sci. USA90:10922-10926; Erb et al., (1994) Proc. Natl. Acad. Sci. USA91:11422-11426; Houghten et al., (1992) Biotechniques 13:412;Jayawickreme et al., (1994) Proc. Natl. Acad. Sci. USA 91:1614-1618;Salmon et al., (1993) Proc. Natl. Acad. Sci. USA 90:11708-11712; PCTPublication No. WO 93/20242, dated Oct. 14, 1993; and Brenner et al.,(1992) Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott et al.,(1990) Science 249:386-390; Devlin et al., (1990) Science, 249:404-406;Christian, et al., (1992)J. Mol. Biol. 227:711-718; Lenstra, (1992) J.Immunol. Meth. 152:149-157; Kay et al., (1993) Gene 128:59-65; and PCTPublication No. WO 94/18318.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058; and Mattheakis etal., (1994) Proc. Natl. Acad. Sci. USA 91:9022-9026.

As used herein, the term “ligand source” can be any compound librarydescribed herein, or tissue extract prepared from various organs in anorganism's system, that can be used to screen for compounds that wouldact as an agonist or antagonist of a Jak 1, Jak 2, Jak3, Stat 1 or Stat2 protein. Screening compound libraries listed herein [also see U.S.Patent Application Publication No. 2005/0009163, which is herebyincorporated by reference in its entirety], in combination with in vivoanimal studies, functional and signaling assays described below can beused to identify Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulatingcompounds that regulate hair growth or treat hair loss disorders.

Screening the libraries can be accomplished by any variety of commonlyknown methods. See, for example, the following references, whichdisclose screening of peptide libraries: Parmley and Smith, (1989) Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, (1990) Science249:386-390; Fowlkes et al., (1992) BioTechniques 13:422-427; Oldenburget al., (1992) Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al.,(1994) Cell 76:933-945; Staudt et al., (1988) Science 241:577-580; Bocket al., (1992) Nature 355:564-566; Tuerk et al., (1992) Proc. Natl.Acad. Sci. USA 89:6988-6992; Ellington et al., (1992) Nature355:850-852; U.S. Pat. Nos. 5,096,815; 5,223,409; and 5,198,346, all toLadner et al.; Rebar et al., (1993) Science 263:671-673; and PCT Pub. WO94/18318.

Small molecule combinatorial libraries can also be generated andscreened. A combinatorial library of small organic compounds is acollection of closely related analogs that differ from each other in oneor more points of diversity and are synthesized by organic techniquesusing multi-step processes. Combinatorial libraries include a vastnumber of small organic compounds. One type of combinatorial library isprepared by means of parallel synthesis methods to produce a compoundarray. A compound array can be a collection of compounds identifiable bytheir spatial addresses in Cartesian coordinates and arranged such thateach compound has a common molecular core and one or more variablestructural diversity elements. The compounds in such a compound arrayare produced in parallel in separate reaction vessels, with eachcompound identified and tracked by its spatial address. Examples ofparallel synthesis mixtures and parallel synthesis methods are providedin U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and its correspondingPCT published patent application WO95/18972, published Jul. 13, 1995 andU.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and its corresponding PCTpublished patent application WO96/22529, which are hereby incorporatedby reference.

In one non-limiting example, non-peptide libraries, such as abenzodiazepine library (see e.g., Bunin et al., (1994) Proc. Natl. Acad.Sci. USA 91:4708-4712), can be screened. Peptoid libraries, such as thatdescribed by Simon et al., (1992) Proc. Natl. Acad. Sci. USA89:9367-9371, can also be used. Another example of a library that can beused, in which the amide functionalities in peptides have beenpermethylated to generate a chemically transformed combinatoriallibrary, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci.USA 91:11138-11142.

Computer modeling and searching technologies permit the identificationof compounds, or the improvement of already identified compounds, thatcan modulate the expression or activity of a Jak 1, Jak 2, Jak3, Stat 1or Stat 2 protein. Having identified such a compound or composition, theactive sites or regions of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2protein can be subsequently identified via examining the sites to whichthe compounds bind. These sites can be ligand binding sites and can beidentified using methods known in the art including, for example, fromthe amino acid sequences of peptides, from the nucleotide sequences ofnucleic acids, or from study of complexes of the relevant compound orcomposition with its natural ligand. In the latter case, chemical orX-ray crystallographic methods can be used to find the active site byfinding where on the factor the complexed ligand is found.

The three dimensional geometric structure of a site, for example that ofa polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene,can be determined by known methods in the art, such as X-raycrystallography, which can determine a complete molecular structure.Solid or liquid phase NMR can be used to determine certainintramolecular distances. Any other experimental method of structuredetermination can be used to obtain partial or complete geometricstructures. The geometric structures can be measured with a complexedligand, natural or artificial, which can increase the accuracy of theactive site structure determined.

Other methods for preparing or identifying peptides that bind to atarget are known in the art. Molecular imprinting, for instance, can beused for the de novo construction of macromolecular structures such aspeptides that bind to a molecule. See, for example, Kenneth J. Shea,Molecular Imprinting of Synthetic Network Polymers: The De Novosynthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2,No. 5, May 1994; Mosbach, (1994) Trends in Biochem. Sci., 19(9); andWulff, G., in Polymeric Reagents and Catalysts (Ford, W. T., Ed.) ACSSymposium Series No. 308, pp 186-230, American Chemical Society (1986).One method for preparing mimics of a Jak 1, Jak 2, Jak3, Stat 1 or Stat2 modulating compound involves the steps of: (i) polymerization offunctional monomers around a known substrate (the template) thatexhibits a desired activity; (ii) removal of the template molecule; andthen (iii) polymerization of a second class of monomers in, the voidleft by the template, to provide a new molecule which exhibits one ormore desired properties which are similar to that of the template. Inaddition to preparing peptides in this manner other binding moleculessuch as polysaccharides, nucleosides, drugs, nucleoproteins,lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and otherbiologically active materials can also be prepared. This method isuseful for designing a wide variety of biological mimics that are morestable than their natural counterparts, because they are prepared by thefree radical polymerization of functional monomers, resulting in acompound with a nonbiodegradable backbone. Other methods for designingsuch molecules include for example drug design based on structureactivity relationships, which require the synthesis and evaluation of anumber of compounds and molecular modeling.

Screening Assays

Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 Modulating Compounds.

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compound can be acompound that affects the activity and/or expression of a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein in vivo and/or in vitro. Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 modulating compounds can be agonists andantagonists of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, and canbe compounds that exert their effect on the activity of a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein via the expression, viapost-translational modifications, or by other means.

Test compounds or agents which bind to a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 protein, and/or have a stimulatory or inhibitory effect on theactivity or the expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2protein, can be identified by two types of assays: (a) cell-based assayswhich utilize cells expressing a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2protein or a variant thereof on the cell surface; or (b) cell-freeassays, which can make use of isolated Jak 1, Jak 2, Jak3, Stat 1 orStat 2 proteins. These assays can employ a biologically active fragmentof a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, full-length proteins,or a fusion protein which includes all or a portion of a polypeptideencoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. A Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein can be obtained from any suitablemammalian species (e.g., human, rat, chick, xenopus, equine, bovine ormurine). The assay can be a binding assay comprising direct or indirectmeasurement of the binding of a test compound. The assay can also be anactivity assay comprising direct or indirect measurement of the activityof a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein. The assay can also bean expression assay comprising direct or indirect measurement of theexpression of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acidsequences or a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2gene. The various screening assays can be combined with an in vivo assaycomprising measuring the effect of the test compound on the symptoms ofa hair loss disorder or disease in a subject (for example, androgeneticalopecia, alopecia areata, alopecia totalis, or alopecia universalis),loss of hair pigmentation in a subject, or even hypotrichosis.

An in vivo assay can also comprise assessing the effect of a testcompound on regulating hair growth in known mammalian models thatdisplay defective or aberrant hair growth phenotypes or mammals thatcontain mutations in the open reading frame (ORF) of nucleic acidsequences comprising a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene thataffects hair growth regulation or hair density, or hair pigmentation. Inone embodiment, controlling hair growth can comprise an induction ofhair growth or density in the subject. Here, the compound's effect inregulating hair growth can be observed either visually via examining theorganism's physical hair growth or loss, or by assessing protein or mRNAexpression using methods known in the art.

Assays for screening test compounds that bind to or modulate theactivity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein can also becarried out. The test compound can be obtained by any suitable means,such as from conventional compound libraries. Determining the ability ofthe test compound to bind to a membrane-bound form of the Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein can be accomplished via coupling the testcompound with a radioisotope or enzymatic label such that binding of thetest compound to the cell expressing a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 protein can be measured by detecting the labeled compound in acomplex. For example, the test compound can be labeled with ³H, ¹⁴C,³⁵S, or ¹²⁵I, either directly or indirectly, and the radioisotope can besubsequently detected by direct counting of radioemmission or byscintillation counting. Alternatively, the test compound can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

Cell-based assays can comprise contacting a cell expressing Jak 1, Jak2, Jak3, Stat 1 or Stat 2 with a test agent and determining the abilityof the test agent to modulate (such as increase or decrease) theactivity or the expression of the membrane-bound molecule. Determiningthe ability of the test agent to modulate the activity of themembrane-bound Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 molecule can beaccomplished by any method suitable for measuring the activity of such amolecule, such as monitoring downstream signaling events (e.g., You etal., Ann N Y Acad Sci. 2008 December; 1150:300-10; Posadas et al.,Expert Rev Clin Immunol. 2009 January; 5(1):9-17; Korhonen et al., BasicClin Pharmacol Toxicol. 2009 April; 104(4):276-84; Vital et al., TherClin Risk Manag. 2006 December; 2(4):365-75; Malek and Castro, Immunity.2010 Aug. 27; 33(2):153-65; Cheng et al., Immunol Rev. 2011 May;241(1):63-76; Lanier, Nat Immunol. 2008 May; 9(5):495-502; Lowell, ColdSpring Harb Perspect Biol. 2011 Mar. 1; 3(3). pii: a002352; Möcsai etal., Nat Rev Immunol. 2010 June; 10(6):387-402; Bradshaw, Cell Signal.2010 August; 22(8):1175-84; Ivanenkov et al., Mini Rev Med Chem. 2011January; 11(1):55-78; Himpe et al., Biofactors. 2009 January-February;35(1):76-81, each of which are incorporated by reference in theirentireties).

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein or the target of a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 protein can be immobilized to facilitatethe separation of complexed from uncomplexed forms of one or both of theproteins. Binding of a test compound to a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 protein or a variant thereof, or interaction of a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein with a target molecule in the presenceand absence of a test compound, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix (for example,glutathione-S-transferase (GST) fusion proteins orglutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) orglutathione derivatized microtiter plates).

A Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, or a variant thereof,can also be immobilized via being bound to a solid support. Non-limitingexamples of suitable solid supports include glass or plastic slides,tissue culture plates, microtiter wells, tubes, silicon chips, orparticles such as beads (including, but not limited to, latex,polystyrene, or glass beads). Any method known in the art can be used toattach a polypeptide (or polynucleotide) corresponding to Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 or a variant thereof, or test compound to a solidsupport, including use of covalent and non-covalent linkages, or passiveabsorption.

The expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein canalso be monitored. For example, regulators of the expression of a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 protein can be identified via contacting acell with a test compound and determining the expression of a proteinencoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell. Theexpression level of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acidsequences in the cell in the presence of the test compound is comparedto the protein or mRNA expression level in the absence of the testcompound. The test compound can then be identified as a regulator of theexpression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein based onthis comparison. For example, when expression of a protein encoded by aJak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2, Jak3, Stat 1or Stat 2 mRNA nucleic acid sequences in the cell is statistically orsignificantly greater in the presence of the test compound than in itsabsence, the test compound is identified as a stimulator/enhancer ofexpression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acidsequences in the cell. The test compound can be said to be a Jak 1, Jak2, Jak3, Stat 1 or Stat 2 modulating compound (such as an agonist).

Alternatively, when expression of a protein encoded by a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 mRNAnucleic acid sequences in the cell is statistically or significantlyless in the presence of the test compound than in its absence, thecompound is identified as an inhibitor of the expression of a proteinencoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell. The testcompound can also be said to be a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2modulating compound (such as an antagonist). The expression level of aprotein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 mRNA nucleic acid sequences in the cell incells can be determined by methods previously described.

For binding assays, the test compound can be a small molecule whichbinds to and occupies the binding site of a polypeptide encoded by a Jak1, Jak 2, Jak3, Stat 1 or Stat 2 gene, or a variant thereof. This canmake the ligand binding site inaccessible to substrate such that normalbiological activity is prevented. Examples of such small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules. In binding assays, either the test compound or a polypeptideencoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can comprise adetectable label, such as a fluorescent, radioisotopic,chemiluminescent, or enzymatic label (for example, alkaline phosphatase,horseradish peroxidase, or luciferase). Detection of a test compoundwhich is bound to a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1or Stat 2 gene can then be determined via direct counting ofradioemmission, by scintillation counting, or by determining conversionof an appropriate substrate to a detectable product.

Determining the ability of a test compound to bind to a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 protein also can be accomplished using real-timeBiamolecular Interaction Analysis (BIA) [McConnell et al., 1992, Science257, 1906-1912; Sjolander, Urbaniczky, 1991, Anal. Chem. 63, 2338-2345].BIA is a technology for studying biospecific interactions in real time,without labeling any of the interactants (for example, BIACORE™).Changes in the optical phenomenon surface plasmon resonance (SPR) can beused as an indication of real-time reactions between biologicalmolecules.

To identify other proteins which bind to or interact with a Jak 1, Jak2, Jak3, Stat 1 or Stat 2 protein and modulate its activity, apolypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene canbe used as a bait protein in a two-hybrid assay or three-hybrid assay(Szabo et al., 1995, Curr. Opin. Struct. Biol. 5, 699-705; U.S. Pat. No.5,283,317), according to methods practiced in the art. The two-hybridsystem is based on the modular nature of most transcription factors,which consist of separable DNA-binding and activation domains.

Functional Assays.

Test compounds can be tested for the ability to increase or decrease theactivity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, or a variantthereof. Activity can be measured after contacting a purified Jak 1, Jak2, Jak3, Stat 1 or Stat 2 protein, a cell membrane preparation, or anintact cell with a test compound. A test compound that decreases theactivity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 90%, at least about 95% or100% is identified as a potential agent for decreasing the activity of aJak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, for example an antagonist.A test compound that increases the activity of a Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 protein by at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 90%, at least about 95% or 100% is identified as a potentialagent for increasing the activity of a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 protein, for example an agonist.

Treatment and Prevention

The invention also provides a method for treating or preventing ahair-loss disorder in a subject. In one embodiment, the method comprisesdetecting the presence of an alteration in a Jak 1, Jak 2, Jak3, Stat 1or Stat 2 gene in a sample from the subject, the presence of thealteration being indicative of a hair-loss disorder, or thepredisposition to a hair-loss disorder, and, administering to thesubject in need a therapeutic treatment against a hair-loss disorder.The therapeutic treatment can be a drug administration (for example, apharmaceutical composition comprising a siRNA directed to a Jak 1, Jak2, Jak3, Stat 1 or Stat 2 nucleic acid). In one embodiment, thetherapeutic molecule to be administered comprises a polypeptide encodedby a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, comprising at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 93%, at least about 95%, at least about 97%, at leastabout 98%, at least about 99%, or 100% of the amino acid sequence of thecorresponding sequence disclosed herein, and exhibits the function ofdecreasing expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat1 or Stat 2 gene. This can restore the capacity to initiate hair growthin cells derived from hair follicles or skin. In another embodiment, thetherapeutic molecule to be administered comprises a nucleic acidsequence comprising a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene thatencodes a polypeptide, comprising at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 93%, atleast about 95%, at least about 97%, at least about 98%, at least about99%, or 100% of the nucleic acid sequence of the corresponding sequencedisclosed herein, and encodes a polypeptide with the function ofdecreasing expression of a protein encoded by a Jak 1, Jak 2, Jak3, Stat1 or Stat 2 gene, thus restoring the capacity to initiate hair growth incells derived from hair follicle cells or skin.

The alteration can be determined at the level of the DNA, RNA, orpolypeptide. Optionally, detection can be determined by performing anoligonucleotide ligation assay, a confirmation based assay, ahybridization assay, a sequencing assay, an allele-specificamplification assay, a microsequencing assay, a melting curve analysis,a denaturing high performance liquid chromatography (DHPLC) assay (forexample, see Jones et al, (2000) Hum Genet., 106(6):663-8), or acombination thereof. In another embodiment, the detection is performedby sequencing all or part of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 geneor by selective hybridization or amplification of all or part of a Jak1, Jak 2, Jak3, Stat 1 or Stat 2 gene. A Jak 1, Jak 2, Jak3, Stat 1 orStat 2 gene specific amplification can be carried out before thealteration identification step.

An alteration in a chromosome region occupied by a Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 gene can be any form of mutation(s), deletion(s),rearrangement(s) and/or insertions in the coding and/or non-codingregion of the locus, alone or in various combination(s). Mutations caninclude point mutations. Insertions can encompass the addition of one orseveral residues in a coding or non-coding portion of the gene locus.Insertions can comprise an addition of between 1 and 50 base pairs inthe gene locus. Deletions can encompass any region of one, two or moreresidues in a coding or non-coding portion of the gene locus, such asfrom two residues up to the entire gene or locus. Deletions can affectsmaller regions, such as domains (introns) or repeated sequences orfragments of less than about 50 consecutive base pairs, although largerdeletions can occur as well. Rearrangement includes inversion ofsequences. The alteration in a chromosome region occupied by a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 gene can result in amino acidsubstitutions, RNA splicing or processing, product instability, thecreation of stop codons, frame-shift mutations, and/or truncatedpolypeptide production. The alteration can result in the production of apolypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene withaltered function, stability, targeting or structure. The alteration canalso cause a reduction, or even an increase in protein expression. Inone embodiment, the alteration in the chromosome region occupied by aJak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene can comprise a point mutation,a deletion, or an insertion in a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2gene or corresponding expression product. In another embodiment, thealteration can be a deletion or partial deletion of a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 gene. The alteration can be determined at thelevel of the DNA, RNA, or polypeptide.

In another embodiment, the method can comprise detecting the presence ofaltered RNA expression. Altered RNA expression includes the presence ofan altered RNA sequence, the presence of an altered RNA splicing orprocessing, or the presence of an altered quantity of RNA. These can bedetected by various techniques known in the art, including sequencingall or part of the RNA or by selective hybridization or selectiveamplification of all or part of the RNA. In a further embodiment, themethod can comprise detecting the presence of altered expression of apolypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene.Altered polypeptide expression includes the presence of an alteredpolypeptide sequence, the presence of an altered quantity ofpolypeptide, or the presence of an altered tissue distribution. Thesecan be detected by various techniques known in the art, including bysequencing and/or binding to specific ligands (such as antibodies).

Various techniques known in the art can be used to detect or quantifyaltered gene or RNA expression or nucleic acid sequences, which include,but are not limited to, hybridization, sequencing, amplification, and/orbinding to specific ligands (such as antibodies). Other suitable methodsinclude allele-specific oligonucleotide (ASO), oligonucleotide ligation,allele-specific amplification, Southern blot (for DNAs), Northern blot(for RNAs), single-stranded conformation analysis (SSCA), PFGE,fluorescent in situ hybridization (FISH), gel migration, clampeddenaturing gel electrophoresis, denaturing HLPC, melting curve analysis,heteroduplex analysis, RNase protection, chemical or enzymatic mismatchcleavage, ELISA, radioimmunoassays (RIA) and immuno-enzymatic assays(IEMA). Some of these approaches (such as SSCA and CGGE) are based on achange in electrophoretic mobility of the nucleic acids, as a result ofthe presence of an altered sequence. According to these techniques, thealtered sequence is visualized by a shift in mobility on gels. Thefragments can then be sequenced to confirm the alteration. Some otherapproaches are based on specific hybridization between nucleic acidsfrom the subject and a probe specific for wild type or altered gene orRNA. The probe can be in suspension or immobilized on a substrate. Theprobe can be labeled to facilitate detection of hybrids. Some of theseapproaches are suited for assessing a polypeptide sequence or expressionlevel, such as Northern blot, ELISA and RIA. These latter require theuse of a ligand specific for the polypeptide, for example, the use of aspecific antibody.

Sequencing.

Sequencing can be carried out using techniques well known in the art,using automatic sequencers. The sequencing can be performed on thecomplete Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or on specificdomains thereof, such as those known or suspected to carry deleteriousmutations or other alterations.

Amplification.

Amplification is based on the formation of specific hybrids betweencomplementary nucleic acid sequences that serve to initiate nucleic acidreproduction. Amplification can be performed according to varioustechniques known in the art, such as by polymerase chain reaction (PCR),ligase chain reaction (LCR), strand displacement amplification (SDA) andnucleic acid sequence based amplification (NASBA). These techniques canbe performed using commercially available reagents and protocols. Usefultechniques in the art encompass real-time PCR, allele-specific PCR, orPCR-SSCP. Amplification usually requires the use of specific nucleicacid primers, to initiate the reaction. Nucleic acid primers useful foramplifying sequences from a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene orlocus are able to specifically hybridize with a portion of a Jak 1, Jak2, Jak3, Stat 1 or Stat 2 gene locus that flank a target region of thelocus, wherein the target region is altered in certain subjects having ahair-loss disorder. In one embodiment, amplification can comprise usingforward and reverse PCR primers comprising nucleotide sequences of thecorresponding sequence disclosed herein.

The invention provides for a nucleic acid primer, wherein the primer canbe complementary to and hybridize specifically to a portion of a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 coding sequence (e.g., gene or RNA)altered in certain subjects having a hair-loss disorder. Primers of theinvention can be specific for altered sequences in a Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 gene or RNA. By using such primers, the detection of anamplification product indicates the presence of an alteration in a Jak1, Jak 2, Jak3, Stat 1 or Stat 2 gene or the absence of such gene.Primers can also be used to identify single nucleotide polymorphisms(SNPs) located in or around a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 genelocus; SNPs can comprise a single nucleotide change, or a cluster ofSNPs in and around a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. Examplesof primers of this invention can be single-stranded nucleic acidmolecules of about 5 to 60 nucleotides in length, or about 8 to about 25nucleotides in length. The sequence can be derived directly from thesequence of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene. Perfectcomplementarity is useful to ensure high specificity; however, certainmismatch can be tolerated. For example, a nucleic acid primer or a pairof nucleic acid primers as described above can be used in a method fordetecting the presence of or a predisposition to a hair-loss disorder ina subject.

Amplification methods include, e.g., polymerase chain reaction, PCR (PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y., 1990 and PCR STRATEGIES, 1995, ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4:560,1989; Landegren, Science 241:1077, 1988; Barringer, Gene 89:117, 1990);transcription amplification (see, e.g., Kwoh, Proc. Natl. Acad. Sci. USA86:1173, 1989); and, self-sustained sequence replication (see, e.g.,Guatelli, Proc. Natl. Acad. Sci. USA 87:1874, 1990); Q Beta replicaseamplification (see, e.g., Smith, J. Clin. Microbiol. 35:1477-1491,1997), automated Q-beta replicase amplification assay (see, e.g., Burg,Mol. Cell. Probes 10:257-271, 1996) and other RNA polymerase mediatedtechniques (e.g., NASBA, Cangene, Mississauga, Ontario); see alsoBerger, Methods Enzymol. 152:307-316, 1987; Sambrook; Ausubel; U.S. Pat.Nos. 4,683,195 and 4,683,202; Sooknanan, Biotechnology 13:563-564, 1995.All the references stated above, an throughout the description, areincorporated by reference in their entireties.

Selective Hybridization.

Hybridization detection methods are based on the formation of specifichybrids between complementary nucleic acid sequences that serve todetect nucleic acid sequence alteration(s). A detection techniqueinvolves the use of a nucleic acid probe specific for wild type oraltered gene or RNA, followed by the detection of the presence of ahybrid. The probe can be in suspension or immobilized on a substrate orsupport (for example, as in nucleic acid array or chips technologies).The probe can be labeled to facilitate detection of hybrids. Forexample, a sample from the subject can be contacted with a nucleic acidprobe specific for a wild type Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 geneor an altered Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, and theformation of a hybrid can be subsequently assessed. In one embodiment,the method comprises contacting simultaneously the sample with a set ofprobes that are specific, respectively, for a wild type Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 gene and for various altered forms thereof. Thus,it is possible to detect directly the presence of various forms ofalterations in a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene in thesample. Also, various samples from various subjects can be treated inparallel.

According to the invention, a probe can be a polynucleotide sequencewhich is complementary to and can specifically hybridize with a (targetportion of a) Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or RNA, and thatis suitable for detecting polynucleotide polymorphisms associated withalleles of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene (or genes) whichpredispose to or are associated with a hair-loss disorder. Useful probesare those that are complementary to a Jak 1, Jak 2, Jak3, Stat 1 or Stat2 gene, RNA, or target portion thereof. Probes can comprisesingle-stranded nucleic acids of between 8 to 1000 nucleotides inlength, for instance between 10 and 800, between 15 and 700, or between20 and 500. Longer probes can be used as well. A useful probe of theinvention is a single stranded nucleic acid molecule of between 8 to 500nucleotides in length, which can specifically hybridize to a region of aJak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or RNA that carries analteration. For example, the probe can be directed to a chromosomeregion occupied by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene.

The sequence of the probes can be derived from the sequences of a Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 gene and RNA as provided herein.Nucleotide substitutions can be performed, as well as chemicalmodifications of the probe. Such chemical modifications can beaccomplished to increase the stability of hybrids (e.g., intercalatinggroups) or to label the probe. Some examples of labels include, withoutlimitation, radioactivity, fluorescence, luminescence, and enzymaticlabeling.

A guide to the hybridization of nucleic acids is found in e.g.,Sambrook, ed., Molecular Cloning: A Laboratory Manual (3^(d) Ed.), Vols.1-3, Cold Spring Harbor Laboratory, 2001; Current Protocols in MolecularBiology, Ausubel, ed. John Wiley & Sons, Inc., New York, 1997;Laboratory Techniques in Biochemistry and Molecular Biology:Hybridization with Nucleic Acid Probes, Part I. Theory and Nucleic AcidPreparation, Tijssen, ed. Elsevier, N.Y., 1993.

Specific Ligand Binding

As discussed herein, alteration in a chromosome region occupied by a Jak1, Jak 2, Jak3, Stat 1 or Stat 2 gene or alteration in expression of aJak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, can also be detected byscreening for alteration(s) in a sequence or expression level of apolypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene.Different types of ligands can be used, such as specific antibodies. Inone embodiment, the sample is contacted with an antibody specific for apolypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene andthe formation of an immune complex is subsequently determined. Variousmethods for detecting an immune complex can be used, such as ELISA,radioimmunoassays (RIA) and immuno-enzymatic assays (IEMA).

For example, an antibody can be a polyclonal antibody, a monoclonalantibody, as well as fragments or derivatives thereof havingsubstantially the same antigen specificity. Fragments include Fab,Fab′2, or CDR regions. Derivatives include single-chain antibodies,humanized antibodies, or poly-functional antibodies. An antibodyspecific for a polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 gene can be an antibody that selectively binds such apolypeptide, namely, an antibody raised against a polypeptide encoded bya Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene or an epitope-containingfragment thereof. Although non-specific binding towards other antigenscan occur, binding to the target polypeptide occurs with a higheraffinity and can be reliably discriminated from non-specific binding. Inone embodiment, the method can comprise contacting a sample from thesubject with an antibody specific for a wild type or an altered form ofa polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene,and determining the presence of an immune complex. Optionally, thesample can be contacted to a support coated with antibody specific forthe wild type or altered form of a polypeptide encoded by a Jak 1, Jak2, Jak3, Stat 1 or Stat 2 gene. In one embodiment, the sample can becontacted simultaneously, or in parallel, or sequentially, with variousantibodies specific for different forms of a polypeptide encoded by aJak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, such as a wild type andvarious altered forms thereof.

Gene Therapy and Protein Replacement Methods

Delivery of nucleic acids into viable cells can be effected ex vivo, insitu, or in vivo by use of vectors, such as viral vectors (e.g.,lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or exvivo by use of physical DNA transfer methods (e.g., liposomes orchemical treatments). Non-limiting techniques suitable for the transferof nucleic acid into mammalian cells in vitro include the use ofliposomes, electroporation, microinjection, cell fusion, DEAE-dextran,and the calcium phosphate precipitation method (See, for example,Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998)).Introduction of a nucleic acid or a gene encoding a polypeptide of theinvention can also be accomplished with extrachromosomal substrates(transient expression) or artificial chromosomes (stable expression).Cells can also be cultured ex vivo in the presence of therapeuticcompositions of the present invention in order to proliferate or toproduce a desired effect on or activity in such cells. Treated cells canthen be introduced in vivo for therapeutic purposes.

Nucleic acids can be inserted into vectors and used as gene therapyvectors. A number of viruses have been used as gene transfer vectors,including papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus(Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992;Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson et al., 1992;Stratford-Perricaudet et al., 1990), vaccinia virus (Moss, 1992),adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990), herpesvirusesincluding HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink etal., 1992; Breakfield and Geller, 1987; Freese et al., 1990), andretroviruses of avian (Biandyopadhyay and Temin, 1984; Petropoulos etal., 1992), murine (Miller, 1992; Miller et al., 1985; Sorge et al.,1984; Mann and Baltimore, 1985; Miller et al., 1988), and human origin(Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990;Buchschacher and Panganiban, 1992). Non-limiting examples of in vivogene transfer techniques include transfection with viral (e.g.,retroviral) vectors (see U.S. Pat. No. 5,252,479, which is incorporatedby reference in its entirety) and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993),incorporated entirely by reference). For example, naked DNA vaccines aregenerally known in the art; see Brower, Nature Biotechnology,16:1304-1305 (1998), which is incorporated by reference in its entirety.Gene therapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see, e.g., U.S. Pat. No.5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994.Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g., retroviral vectors, the pharmaceutical preparation caninclude one or more cells that produce the gene delivery system.

For reviews of gene therapy protocols and methods see Anderson et al.,Science 256:808-813 (1992); U.S. Pat. Nos. 5,252,479, 5,747,469,6,017,524, 6,143,290, 6,410,010 6,511,847; and U.S. ApplicationPublication Nos. 2002/0077313 and 2002/00069, which are all herebyincorporated by reference in their entireties. For additional reviews ofgene therapy technology, see Friedmann, Science, 244:1275-1281 (1989);Verma, Scientific American: 68-84 (1990); Miller, Nature, 357: 455-460(1992); Kikuchi et al., J Dermatol Sci. 2008 May; 50(2):87-98; Isaka etal., Expert Opin Drug Deliv. 2007 September; 4(5):561-71; Jager et al.,Curr Gene Ther. 2007 August; 7(4):272-83; Waehler et al., Nat Rev Genet.2007 August; 8(8):573-87; Jensen et al., Ann Med. 2007; 39(2):108-15;Herweijer et al., Gene Ther. 2007 January; 14(2):99-107; Eliyahu et al.,Molecules, 2005 Jan. 31; 10(1):34-64; and Altaras et al., Adv BiochemEng Biotechnol. 2005; 99:193-260, all of which are hereby incorporatedby reference in their entireties.

Protein replacement therapy can increase the amount of protein byexogenously introducing wild-type or biologically functional protein byway of infusion. A replacement polypeptide can be synthesized accordingto known chemical techniques or can be produced and purified via knownmolecular biological techniques. Protein replacement therapy has beendeveloped for various disorders. For example, a wild-type protein can bepurified from a recombinant cellular expression system (e.g., mammaliancells or insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.;U.S. Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No.6,461,609 to Calhoun et al.; U.S. Pat. No. 6,210,666 to Miyamura et al.;U.S. Pat. No. 6,083,725 to Selden et al.; U.S. Pat. No. 6,451,600 toRasmussen et al.; U.S. Pat. No. 5,236,838 to Rasmussen et al. and U.S.Pat. No. 5,879,680 to Ginns et al.), human placenta, or animal milk (seeU.S. Pat. No. 6,188,045 to Reuser et al.), or other sources known in theart. After the infusion, the exogenous protein can be taken up bytissues through non-specific or receptor-mediated mechanism.

A polypeptide encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene canalso be delivered in a controlled release system. For example, thepolypeptide can be administered using intravenous infusion, animplantable osmotic pump, a transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump can be used (see is Langer,supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald etal., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)). In another embodiment, polymeric materials can be used (seeMedical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York(1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990)).

Pharmaceutical Compositions and Administration for Therapy

Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 modulating compounds of the invention can beadministered to the subject once (e.g., as a single injection ordeposition). Alternatively, Jak 1, Jak 2, Jak3, Stat 1 or Stat 2proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compoundscan be administered once or twice daily to a subject in need thereof fora period of from about two to about twenty-eight days, or from aboutseven to about ten days. Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteinsand Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds can alsobe administered once or twice daily to a subject for a period of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combinationthereof. Furthermore, Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins andJak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds of theinvention can be co-administrated with another therapeutic. Where adosage regimen comprises multiple administrations, the effective amountof the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 modulating compounds administered to the subjectcan comprise the total amount of gene product administered over theentire dosage regimen.

Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 modulating compounds can be administered to a subjectby any means suitable for delivering the Jak 1, Jak 2, Jak3, Stat 1 orStat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulatingcompounds to cells of the subject, such as the dermis, epidermis, dermalpapilla cells, or hair follicle cells. For example, Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 proteins and Jak 1, Jak 2, Jak3, Stat 1 or Stat 2modulating compounds can be administered by methods suitable totransfect cells. Transfection methods for eukaryotic cells are wellknown in the art, and include direct injection of the nucleic acid intothe nucleus or pronucleus of a cell; electroporation; liposome transferor transfer mediated by lipophilic materials; receptor mediated nucleicacid delivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

The compositions of this invention can be formulated and administered toreduce the symptoms associated with a hair-loss disorder by any meansthat produces contact of the active ingredient with the agent's site ofaction in the body of a subject, such as a human or animal (e.g., a dog,cat, or horse). They can be administered by any conventional meansavailable for use in conjunction with pharmaceuticals, either asindividual therapeutic active ingredients or in a combination oftherapeutic active ingredients. They can be administered alone, but aregenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

A therapeutically effective dose of Jak 1, Jak 2, Jak3, Stat 1 or Stat 2modulating compounds can depend upon a number of factors known to thoseor ordinary skill in the art. The dose(s) of the Jak 1, Jak 2, Jak3,Stat 1 or Stat 2 modulating compounds can vary, for example, dependingupon the identity, size, and condition of the subject or sample beingtreated, further depending upon the route by which the composition is tobe administered, if applicable, and the effect which the practitionerdesires the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds tohave upon the nucleic acid or polypeptide of the invention. Theseamounts can be readily determined by a skilled artisan. Any of thetherapeutic applications described herein can be applied to any subjectin need of such therapy, including, for example, a mammal such as a dog,a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or ahuman.

Pharmaceutical compositions for use in accordance with the invention canbe formulated in conventional manner using one or more physiologicallyacceptable carriers or excipients. The therapeutic compositions of theinvention can be formulated for a variety of routes of administration,including systemic and topical or localized administration. Techniquesand formulations generally can be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. (20^(th) Ed., 2000), theentire disclosure of which is herein incorporated by reference. Forsystemic administration, an injection is useful, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the therapeutic compositions of the invention can beformulated in liquid solutions, for example in physiologicallycompatible buffers such as Hank's solution or Ringer's solution. Inaddition, the therapeutic compositions can be formulated in solid formand redissolved or suspended immediately prior to use. Lyophilized formsare also included. Pharmaceutical compositions of the present inventionare characterized as being at least sterile and pyrogen-free. Thesepharmaceutical formulations include formulations for human andveterinary use.

According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theactive compound can be used. Supplementary active compounds can also beincorporated into the compositions.

The invention also provides for a kit that comprises a pharmaceuticallyacceptable carrier and a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulatingcompound identified using the screening assays of the invention packagedwith instructions for use. For modulators that are antagonists of theactivity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein, or whichreduce the expression of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 protein,the instructions would specify use of the pharmaceutical composition forpromoting the loss of hair on the body surface of a mammal (for example,arms, legs, bikini area, face).

For Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulating compounds that areagonists of the activity of a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2protein or increase the expression of one or more proteins encoded byJak 1, Jak 2, Jak3, Stat 1 or Stat 2 genes, the instructions wouldspecify use of the pharmaceutical composition for regulating hairgrowth. In one embodiment, the instructions would specify use of thepharmaceutical composition for the treatment of hair loss disorders. Ina further embodiment, the instructions would specify use of thepharmaceutical composition for restoring hair pigmentation. For example,administering an agonist can reduce hair graying in a subject.

A pharmaceutical composition containing a Jak 1, Jak 2, Jak3, Stat 1 orStat 2 modulating compound can be administered in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed herein. Such pharmaceutical compositions can comprise, forexample antibodies directed to polypeptides encoded by genes comprisinga Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene, or variants thereof, oragonists and antagonists of a polypeptide encoded by a Jak 1, Jak 2,Jak3, Stat 1 or Stat 2 gene. The compositions can be administered aloneor in combination with at least one other agent, such as a stabilizingcompound, which can be administered in any sterile, biocompatiblepharmaceutical carrier including, but not limited to, saline, bufferedsaline, dextrose, and water. The compositions can be administered to apatient alone, or in combination with other agents, drugs or hormones.

Sterile injectable solutions can be prepared by incorporating the Jak 1,Jak 2, Jak3, Stat 1 or Stat 2 modulating compound (e.g., a polypeptideor antibody) in the required amount in an appropriate solvent with oneor a combination of ingredients enumerated herein, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of useful preparation methods arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In some embodiments, the Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 modulatingcompound can be applied via transdermal delivery systems, which slowlyreleases the active compound for percutaneous absorption. Permeationenhancers can be used to facilitate transdermal penetration of theactive factors in the conditioned media. Transdermal patches aredescribed in for example, U.S. Pat. Nos. 5,407,713; 5,352,456;5,332,213; 5,336,168; 5,290,561; 5,254,346; 5,164,189; 5,163,899;5,088,977; 5,087,240; 5,008,110; and 4,921,475.

Various routes of administration and various sites of cell implantationcan be utilized, such as, subcutaneous or intramuscular, in order tointroduce the aggregated population of cells into a site of preference.Once implanted in a subject (such as a mouse, rat, or human), theaggregated cells can then stimulate the formation of a hair follicle andthe subsequent growth of a hair structure at the site of introduction.In another embodiment, transfected cells (for example, cells expressinga protein encoded by a Jak 1, Jak 2, Jak3, Stat 1 or Stat 2 gene areimplanted in a subject to promote the formation of hair follicles withinthe subject. In further embodiments, the transfected cells are cellsderived from the end bulb of a hair follicle (such as dermal papillacells or dermal sheath cells). Aggregated cells (for example, cellsgrown in a hanging drop culture) or transfected cells (for example,cells produced as described herein) maintained for 1 or more passagescan be introduced (or implanted) into a subject (such as a rat, mouse,dog, cat, human, and the like).

“Subcutaneous” administration can refer to administration just beneaththe skin (i.e., beneath the dermis). Generally, the subcutaneous tissueis a layer of fat and connective tissue that houses larger blood vesselsand nerves. The size of this layer varies throughout the body and fromperson to person. The interface between the subcutaneous and musclelayers can be encompassed by subcutaneous administration.

This mode of administration can be feasible where the subcutaneous layeris sufficiently thin so that the factors present in the compositions canmigrate or diffuse from the locus of administration and contact the hairfollicle cells responsible for hair formation. Thus, where intradermaladministration is utilized, the bolus of composition administered islocalized proximate to the subcutaneous layer.

Administration of the cell aggregates (such as DP or DS aggregates) isnot restricted to a single route, but can encompass administration bymultiple routes. For instance, exemplary administrations by multipleroutes include, among others, a combination of intradermal andintramuscular administration, or intradermal and subcutaneousadministration. Multiple administrations can be sequential orconcurrent. Other modes of application by multiple routes will beapparent to the skilled artisan.

In other embodiments, this implantation method will be a one-timetreatment for some subjects. In further embodiments of the invention,multiple cell therapy implantations will be required. In someembodiments, the cells used for implantation will generally besubject-specific genetically engineered cells. In another embodiment,cells obtained from a different species or another individual of thesame species can be used. Thus, using such cells can requireadministering an immunosuppressant to prevent rejection of the implantedcells. Such methods have also been described in United States PatentApplication Publication 2004/0057937 and PCT application publication WO2001/32840, and are hereby incorporated by reference.

Inhibitors

Cytokines are produced to activate neighboring cells to communicatedanger signals to one another and spread and amplify the inflammatoryresponse. Over the years, it was learned how to both neutralize thesecytokines with blocking antibodies and inhibit their signaling inresponding cells by small molecule protein tyrosine kinase inhibitorsFDA approved drugs exist for both approaches, e.g., IL-2 and TNFblocking antibodies and the small orally available molecule Gleevec™that blocks cytokine signaling. Whenever possible, the center willpursue topical small molecule formulations that should improve efficacywhile limiting systemic toxicity (improved therapeutic indexes)encouraging clinical evaluation in AA of other small molecule inhibitorsin the biopharmaceutical pipeline.

To block signaling of cytokine receptors, topical/oral JAK1/2 inhibitor(Incyte), which has already demonstrated safety and efficacy in patientswith psoriasis and RA, can be used. Similarly, topical and/or oral JAK3inhibitors as described herein (e.g., tofacitinib (CP690550), R348 andVX-509), which have demonstrated preliminary safety and efficacy inpatients with RA, can be used.

The inhibitors can comprise peptides (such as antibodies or fragmentsthereof), small molecules, nucleic acids (such as siRNA or antisenseRNA), or other agents) that can bind to a polypeptide molecule encodedby a gene of interest and/or molecules that have an inhibitory effect onthe biological activity of a protein of interest or its expression.

As used herein, a “Jak 1 inhibitor” refers to a compound that interactswith a Jak 1 gene or a Jak 1 protein or polypeptide and inhibits itsactivity and/or its expression. The compound can decrease the activityor expression of a protein encoded by Jak 1.

In one embodiment, a Jak 1 inhibitor can be a peptide fragment thatbinds a protein comprising SEQ ID NO: 1. For example, the fragment canencompass any portion of at least about 8 consecutive amino acids of SEQID NO: 1. The fragment can comprise at least about 10 consecutive aminoacids, at least about 20 consecutive amino acids, at least about 30consecutive amino acids, at least about 40 consecutive amino acids, atleast about 50 consecutive amino acids, at least about 60 consecutiveamino acids, or at least about 75 consecutive amino acids of SEQ IDNO: 1. Fragments include all possible amino acid lengths between andincluding about 8 and about 100 amino acids, for example, lengthsbetween about 10 and about 100 amino acids, between about 15 and about100 amino acids, between about 20 and about 100 amino acids, betweenabout 35 and about 100 amino acids, between about 40 and about 100 aminoacids, between about 50 and about 100 amino acids, between about 70 andabout 100 amino acids, between about 75 and about 100 amino acids, orbetween about 80 and about 100 amino acids. These peptide fragments canbe obtained commercially or synthesized via liquid phase or solid phasesynthesis methods (Atherton et al., (1989) Solid Phase PeptideSynthesis: a Practical Approach. IRL Press, Oxford, England).

As used herein, a “Jak 2 inhibitor” refers to a compound that interactswith a Jak 2 gene or a Jak 2 protein or polypeptide and inhibits itsactivity and/or its expression. The compound can decrease the activityor expression of a protein encoded by Jak 2.

In one embodiment, a Jak 2 inhibitor can be a peptide fragment thatbinds a protein comprising SEQ ID NO: 3. For example, the fragment canencompass any portion of at least about 8 consecutive amino acids of SEQID NO: 3. The fragment can comprise at least about 10 consecutive aminoacids, at least about 20 consecutive amino acids, at least about 30consecutive amino acids, at least about 40 consecutive amino acids, atleast about 50 consecutive amino acids, at least about 60 consecutiveamino acids, or at least about 75 consecutive amino acids of SEQ ID NO:3. Fragments include all possible amino acid lengths between andincluding about 8 and about 100 amino acids, for example, lengthsbetween about 10 and about 100 amino acids, between about 15 and about100 amino acids, between about 20 and about 100 amino acids, betweenabout 35 and about 100 amino acids, between about 40 and about 100 aminoacids, between about 50 and about 100 amino acids, between about 70 andabout 100 amino acids, between about 75 and about 100 amino acids, orbetween about 80 and about 100 amino acids. These peptide fragments canbe obtained commercially or synthesized via liquid phase or solid phasesynthesis methods (Atherton et al., (1989) Solid Phase PeptideSynthesis: a Practical Approach. IRL Press, Oxford, England).

As used herein, a “Jak 3 inhibitor” can be a compound that interactswith a Jak 3 gene, or a Jak 3 protein or polypeptide, and inhibits itsactivity and/or its expression. The compound can decrease the activityor expression of a protein encoded by Jak 3. In one embodiment, a Jak3inhibitor can be a Jak3 modulating compound.

In one embodiment, a Jak 3 inhibitor can be a peptide fragment thatbinds a protein comprising SEQ ID NO: 109. For example, the fragment canencompass any portion of about 8 consecutive amino acids of SEQ ID NO:109. The fragment can comprise about 10 consecutive amino acids, about20 consecutive amino acids, about 30 consecutive amino acids, about 40consecutive amino acids, about 50 consecutive amino acids, about 60consecutive amino acids, or about 75 consecutive amino acids of SEQ IDNO: 109. Fragments include all possible amino acid lengths between andincluding about 8 and about 100 amino acids, for example, lengthsbetween about 10 and about 100 amino acids, between about 15 and about100 amino acids, between about 20 and about 100 amino acids, betweenabout 35 and about 100 amino acids, between about 40 and about 100 aminoacids, between about 50 and about 100 amino acids, between about 70 andabout 100 amino acids, between about 75 and about 100 amino acids, orbetween about 80 and about 100 amino acids. These peptide fragments canbe obtained commercially or synthesized via liquid phase or solid phasesynthesis methods (Atherton et al., (1989) Solid Phase PeptideSynthesis: a Practical Approach. IRL Press, Oxford, England).

An inhibitor of the invention can be a protein, such as an antibody(monoclonal, polyclonal, humanized, chimeric, or fully human), or abinding fragment thereof, directed against a polypeptide encoded by SEQID NO: 109. An antibody fragment can be a form of an antibody other thanthe full-length form and includes portions or components that existwithin full-length antibodies, in addition to antibody fragments thathave been engineered. Antibody fragments can include, but are notlimited to, single chain Fv (scFv), diabodies, Fv, and (Fab′)₂,triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR's, variableregions, tetrabodies, bifunctional hybrid antibodies, framework regions,constant regions, and the like (see, Maynard et al., (2000) Ann. Rev.Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402).Antibodies can be obtained commercially, custom generated, orsynthesized against an antigen of interest according to methodsestablished in the art (see Roland E. Kontermann and Stefan Dibel(editors), Antibody Engineering, Vol. I & II, (2010) 2^(nd) ed.,Springer; Antony S. Dimitrov (editor), Therapeutic Antibodies: Methodsand Protocols (Methods in Molecular Biology), (2009), Humana Press;Benny Lo (editor) Antibody Engineering: Methods and Protocols (Methodsin Molecular Biology), (2004) Humana Press, each of which are herebyincorporated by reference in their entireties).

As used herein, a “Stat 1 inhibitor” refers to a compound that interactswith a Stat 1 gene or a Stat 1 protein or polypeptide and inhibits itsactivity and/or its expression. The compound can decrease the activityor expression of a protein encoded by Stat 1.

In one embodiment, a Stat 1 inhibitor can be a peptide fragment thatbinds a protein comprising SEQ ID NO: 5. For example, the fragment canencompass any portion of at least about 8 consecutive amino acids of SEQID NO: 5. The fragment can comprise at least about 10 consecutive aminoacids, at least about 20 consecutive amino acids, at least about 30consecutive amino acids, at least about 40 consecutive amino acids, atleast about 50 consecutive amino acids, at least about 60 consecutiveamino acids, or at least about 75 consecutive amino acids of SEQ ID NO:5. Fragments include all possible amino acid lengths between andincluding about 8 and about 100 amino acids, for example, lengthsbetween about 10 and about 100 amino acids, between about 15 and about100 amino acids, between about 20 and about 100 amino acids, betweenabout 35 and about 100 amino acids, between about 40 and about 100 aminoacids, between about 50 and about 100 amino acids, between about 70 andabout 100 amino acids, between about 75 and about 100 amino acids, orbetween about 80 and about 100 amino acids. These peptide fragments canbe obtained commercially or synthesized via liquid phase or solid phasesynthesis methods (Atherton et al., (1989) Solid Phase PeptideSynthesis: a Practical Approach. IRL Press, Oxford, England).

As used herein, a “Stat 2 inhibitor” refers to a compound that interactswith a Stat 2 gene or a Stat 2 protein or polypeptide and inhibits itsactivity and/or its expression. The compound can decrease the activityor expression of a protein encoded by Stat 2.

In one embodiment, a Stat 2 inhibitor can be a peptide fragment thatbinds a protein comprising SEQ ID NO: 7. For example, the fragment canencompass any portion of at least about 8 consecutive amino acids of SEQID NO: 7. The fragment can comprise at least about 10 consecutive aminoacids, at least about 20 consecutive amino acids, at least about 30consecutive amino acids, at least about 40 consecutive amino acids, atleast about 50 consecutive amino acids, at least about 60 consecutiveamino acids, or at least about 75 consecutive amino acids of SEQ ID NO:7. Fragments include all possible amino acid lengths between andincluding about 8 and about 100 amino acids, for example, lengthsbetween about 10 and about 100 amino acids, between about 15 and about100 amino acids, between about 20 and about 100 amino acids, betweenabout 35 and about 100 amino acids, between about 40 and about 100 aminoacids, between about 50 and about 100 amino acids, between about 70 andabout 100 amino acids, between about 75 and about 100 amino acids, orbetween about 80 and about 100 amino acids. These peptide fragments canbe obtained commercially or synthesized via liquid phase or solid phasesynthesis methods (Atherton et al., (1989) Solid Phase PeptideSynthesis: a Practical Approach. IRL Press, Oxford, England).

As used herein, a “Jak/Stat inhibitor” refers to a compound thatinteracts with a Jak1/Jak2/Jak3/Stat1/Stat2 gene or aJak1/Jak2/Jak3/Stat1/Stat2 protein or polypeptide and inhibits itsactivity and/or its expression. The compound can decrease the activityor expression of a protein encoded by Jak1/Jak2/Jak3/Stat1/Stat2.

An inhibitor of the invention can be a protein, such as an antibody(monoclonal, polyclonal, humanized, chimeric, or fully human), or abinding fragment thereof, directed against a polypeptide encoded by thecorresponding sequence disclosed herein. An antibody fragment can be aform of an antibody other than the full-length form and includesportions or components that exist within full-length antibodies, inaddition to antibody fragments that have been engineered. Antibodyfragments can include, but are not limited to, single chain Fv (scFv),diabodies, Fv, and (Fab′)₂, triabodies, Fc, Fab, CDR1, CDR2, CDR3,combinations of CDR's, variable regions, tetrabodies, bifunctionalhybrid antibodies, framework regions, constant regions, and the like(see, Maynard et al., (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson(1998) Curr. Opin. Biotechnol. 9:395-402). Antibodies can be obtainedcommercially, custom generated, or synthesized against an antigen ofinterest according to methods established in the art (Janeway et al.,(2001) Immunobiology, 5th ed., Garland Publishing).

An inhibitor of the invention can also be a small molecule that binds toa protein and disrupts its function. Small molecules are a diverse groupof synthetic and natural substances generally having low molecularweights. They can be isolated from natural sources (for example, plants,fungi, microbes and the like), are obtained commercially and/oravailable as libraries or collections, or synthesized. Candidate smallmolecules that modulate a protein can be identified via in silicoscreening or high-through-put (HTP) screening of combinatoriallibraries. Most conventional pharmaceuticals, such as aspirin,penicillin, and many chemotherapeutics, are small molecules, can beobtained commercially, can be chemically synthesized, or can be obtainedfrom random or combinatorial libraries (Werner et al., (2006) BriefFunct. Genomic Proteomic 5(1):32-6). In some embodiments, the agent is asmall molecule that binds, interacts, or associates with a targetprotein or RNA. Such a small molecule can be an organic molecule that,when the target is an intracellular target, is capable of penetratingthe lipid bilayer of a cell to interact with the target. Small moleculesinclude, but are not limited to, toxins, chelating agents, metals, andmetalloid compounds. Small molecules can be attached or conjugated to atargeting agent so as to specifically guide the small molecule to aparticular cell.

Pharmaceutical Compositions and Administration for Therapy

An inhibitor or agonist of the invention can be incorporated intopharmaceutical compositions suitable for administration, for example theinhibitor and a pharmaceutically acceptable carrier.

According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theactive compound can be used. Supplementary active compounds can also beincorporated into the compositions.

Any of the therapeutic applications described herein can be applied toany subject in need of such therapy, including, for example, a mammalsuch as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, asheep, a goat, or a human.

A pharmaceutical composition of the invention can be administered inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed herein. Such pharmaceutical compositionscan comprise, for example antibodies directed to polypeptides. Thecompositions can be administered alone or in combination with at leastone other agent, such as a stabilizing compound, which can beadministered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions can be administered to a patient alone, or incombination with other agents, drugs or hormones.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, a pharmaceutically acceptable polyol like glycerol,propylene glycol, liquid polyetheylene glycol, and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it can be useful to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theinhibitor (e.g., a polypeptide or antibody or small molecule) or agonistof the invention in the required amount in an appropriate solvent withone or a combination of ingredients enumerated herein, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated herein. In the case of sterile powders for thepreparation of sterile injectable solutions, examples of usefulpreparation methods are vacuum drying and freeze-drying which yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The dosage administered can be a therapeutically effective amount of thecomposition sufficient to result in amelioration of symptoms of alopeciaareata, and can vary depending upon known factors such as thepharmacodynamic characteristics of the active ingredient and its modeand route of administration; age, sex, health and weight of therecipient; nature and extent of symptoms; kind of concurrent treatment,frequency of treatment and the effect desired.

In some embodiments, the effective amount of the administered Jak3modulating compound (e.g., a compound described herein that is directedto Jak3, such as a small molecule JAK3 inhibitor) is about 0.0001 μg/kgbody weight, about 0.00025 μg/kg body weight, about 0.0005 μg/kg bodyweight, about 0.00075 μg/kg body weight, about 0.001 μg/kg body weight,about 0.0025 μg/kg body weight, about 0.005 μg/kg body weight, about0.0075 μg/kg body weight, about 0.01 μg/kg body weight, about 0.025μg/kg body weight, about 0.05 μg/kg body weight, about 0.075 μg/kg bodyweight, about 0.1 μg/kg body weight, about 0.25 μg/kg body weight, about0.5 μg/kg body weight, about 0.75 μg/kg body weight, about 1 μg/kg bodyweight, about 5 μg/kg body weight, about 10 μg/kg body weight, about 25μg/kg body weight, about 50 μg/kg body weight, about 75 μg/kg bodyweight, about 100 μg/kg body weight, about 150 μg/kg body weight, about200 μg/kg body weight, about 250 μg/kg body weight, about 300 μg/kg bodyweight, about 350 μg/kg body weight, about 400 μg/kg body weight, about450 μg/kg body weight, about 500 μg/kg body weight, about 550 μg/kg bodyweight, about 600 μg/kg body weight, about 650 μg/kg body weight, about700 μg/kg body weight, about 750 μg/kg body weight, about 800 μg/kg bodyweight, about 850 μg/kg body weight, about 900 μg/kg body weight, about950 μg/kg body weight, about 1000 μg/kg body weight, about 2000 μg/kgbody weight, about 3000 μg/kg body weight, about 4000 μg/kg body weight,about 5000 μg/kg body weight, about 6000 μg/kg body weight, about 7000μg/kg body weight, about 8000 μg/kg body weight, about 95000 μg/kg bodyweight, or about 10,000 μg/kg body weight.

In some embodiments, the effective amount of the administered Jak3modulating compound (e.g., a small molecule JAK3 inhibitor) is about 1mg/kg body weight, about 1.5 mg/kg body weight, about 2 mg/kg bodyweight, about 2.5 mg/kg body weight, about 3 mg/kg body weight, about3.5 mg/kg body weight, about 4 mg/kg body weight, about 4.5 mg/kg bodyweight, about 5 mg/kg body weight, about 5.5 mg/kg body weight, about 6mg/kg body weight, about 6.5 mg/kg body weight, about 7 mg/kg bodyweight, about 7.5 mg/kg body weight, about 8 mg/kg body weight, about9.5 mg/kg body weight, about 10 mg/kg body weight, about 10.5 mg/kg bodyweight, about 11.0 mg/kg body weight, about 11.5 mg/kg body weight,about 12 mg/kg body weight, about 12.5 mg/kg body weight, about 13 mg/kgbody weight, about 13.5 mg/kg body weight, about 14 mg/kg body weight,about 14.5 mg/kg body weight, about 15 mg/kg body weight, about 15.5mg/kg body weight, about 16 mg/kg body weight, about 16.5 mg/kg bodyweight, about 17 mg/kg body weight, about 17.5 mg/kg body weight, about18 mg/kg body weight, about 19.5 mg/kg body weight, about 20 mg/kg bodyweight, about 21.5 mg/kg body weight, about 22 mg/kg body weight, about22.5 mg/kg body weight, about 23 mg/kg body weight, about 23.5 mg/kgbody weight, about 24 mg/kg body weight, about 24.5 mg/kg body weight,about 25 mg/kg body weight, about 25.5 mg/kg body weight, about 26 mg/kgbody weight, about 26.5 mg/kg body weight, about 27 mg/kg body weight,about 27.5 mg/kg body weight, about 28 mg/kg body weight, about 29.5mg/kg body weight, or about 30 mg/kg body weight.

In other embodiments, the administered Jak3 modulating compound (e.g., acompound described herein that is directed to Jak3, such as a smallmolecule JAK3 inhibitor), can be administered as a topical cream. Insome embodiments, the effective amount of the administered Jak3modulating compound (such as a small molecule JAK3 inhibitor) is presentat a concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%,about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%,about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%,about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%,about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%,about 2.9%, or about 3.0%. In some embodiments, the effective amount ofthe administered Jak3 modulating compound (such as a small molecule JAK3inhibitor) is present at a concentration of about 3.5%, about 4%, about4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%,about 8%, about 8.5%, about 9%, about 9.5%, or of about 10%.

In some embodiments, the Jak3 modulating compound (e.g., a compounddescribed herein that is directed to Jak3, such as a small molecule JAK3inhibitor), can be co-administered with a second JAK inhibitor (such asa JAK1/2 inhibitor). In one embodiment, the JAK1/2 inhibitor can beadministered as a topical cream. In some embodiments, the effectiveamount of the administered small molecule JAK1/2 inhibitor is present ata concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about2.9%, or about 3.0%. In some embodiments, the effective amount of theadministered JAK1/2 inhibitor is present at a concentration of about3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%,about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or ofabout 10%.

Toxicity and therapeutic efficacy of therapeutic compositions of thepresent invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Therapeutic agentsthat exhibit large therapeutic indices are useful. Therapeuticcompositions that exhibit some toxic side effects can be used.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

Example 1

Interferon γ has been shown to be a target in murine AA (see Nakamura,et al. 2008, Am J Pathol, 172(3): 650-658; see also Freyschmidt-Paul etal., Br J Dermatol., 155(3):515-521; see also Gilhar et al., JournalInvest. Dermatol., 124(1):288-289). In human AA, AA PBMCs show Th1skewing, AA skin shows IFN signature and GWAS genes (SOCS/IFN-g/). butalso IL-2/6/13/21/26.

The interferon γ pathway (FIG. 7) induces NKG2DL in target HF cells,enhances antigen presentation by DCs, promotes Th1 cellular autoimmunityand augments NK and CTL mediated-cytolysis.

JAK1/2 inhibitors in clinical development include a) INCB018424, topicaland oral; 5 nM activity (Incyte); b) CEP-701 (Cephalon); and c)TG101348.

Vehicle Active Medra Preferred Term (n = 50) (n = 149) Application siteirritation, pruritus  7 (14.0) 14 (9.3)  Nasopharyngitis 1 (2.0) 10(6.7)  Upper respiratory tract infection 3 (6.0) 10 (6.7)  Sinusitis 1(2.0) 6 (4.0) Back pain 0 (0.0) 5 (3.4) Headache 0 (0.0) 4 (2.7)Influenza 2 (4.0) 4 (2.7) Abdominal pain 0 (0.0) 3 (2.0)

-   -   Six SAEs, none treatment related: prostate ca, prolonged        hospitalization for cholecystitis, cholelithiasis (1.5%);        herniated disc repair, coronary occlusion₁₆ with hx ASHD (0.5%).        CVA (vehicle)        Table 4. INCB18424-203: Treatment-Emergent Adverse Events in        Descending Order (all casualties) (oral delivery is considered        an acceptable profile)

Clinical programs in AA include the following:

-   Mild-Moderate AA: Phase 2/3 Psoriasis and oral Phase 3    Myelofibrosis, using a topical JAK inhibitor

A population-based cohort evaluated cases ascertained through NationalAlopecia Areata Registry vs. controls ascertained through New York CityCancer project. They were matched for Northern European ancestry usingAIMS.

-   Cases: n=1080 (Alopecia Universalis=482; Alopecia Totalis=120;    Alopecia Areata Transient=176; Alopecia Areata Patchy=302)-   Controls: n=1053

FIG. 9 presents a Genomewide Association Study (GWAS) in alopeciaareata.

TABLE 5 List of Genes implicated in AA. Stongest association MaximumRegion Gene Function (pvalue) odds ratio  2q33.2 CTLA4 T-cellproliferation 3.55 × 10⁻¹³ 1.44 ICOS T-cell proliferation 4.33 × 10⁻⁰⁸1.32  4q27 IL21/IL2 T-, B- and NK-cell 4.27 × 10⁻⁰⁸ 1.34 proliferation 6q25.1 ULBP6 NKG2D activating 4.49 × 10⁻¹⁹ 1.65 ligand ULBP3 NKG2Dactivating 4.43 × 10⁻¹⁷ 1.52 ligand  9q31.1 STX17 premature hair 3.60 ×10⁻⁰⁷ 1.33 graying 10p15.1 IL2RA T-cell proliferation 1.74 × 10⁻¹² 1.41(CD25) 11q13 PRDX5 antioxidant enzyme 4.14 × 10⁻⁰⁷ 1.33 12q13 Eos(IKZF4) T-cell proliferation 3.21 × 10⁻⁰⁸ 1.34 ERBB3 epidermal growth1.27 × 10⁻⁰⁷ 1.34 factor receptor  6p21.32 MICA NKG2D activating 1.19 ×10⁻⁰⁷ 1.44 (HLA) ligand NOTCH4 T-cell differentiation 1.03 × 10⁻⁰⁸ 1.61BTNL2 T-cell differentiation 2.11 × 10⁻²⁶ 2.70 HLA-DRA Antigenpresentation 2.93 × 10⁻³¹ 2.62 HLA-DQA1 Antigen presentation 3.60 ×10⁻¹⁷ 2.15 HLA-DQA2 Antigen presentation 1.38 × 10⁻³⁵ 5.43 HLA-DQB2Antigen presentation 1.73 × 10⁻¹³ 1.60 HLA-DOB Antigen presentation 2.07× 10⁻⁰⁸ 1.72

TABLE 6 Common Cause Model of Skin Autoimmune Diseases. See also Jin etal., 2010, NEJM, 362:1686-1697. Stongest Involved in other associationMaximum autoimmune Region Gene (pvalue) odds ratio disease  2q33.2 CTLA43.55 × 10⁻¹³ 1.44 T1D, RA, CeD, MS, SLE, GD ICOS 4.33 × 10⁻⁰⁸ 1.32 T1D,RA, CeD, MS, SLE, GD  4q27 IL21/IL2 4.27 × 10⁻⁰⁸ 1.34 ● T1D, RA, CeD, PS 6q25.1 ULBP6 4.49 × 10⁻¹⁹ 1.65 none ULBP3 4.43 × 10⁻¹⁷ 1.52 none 9q31.1 STX17 3.60 × 10⁻⁰⁷ 1.33 none 10p15.1 IL2RA 1.74 × 10⁻¹² 1.41 ○T1D, MS, GD, (CD25) GV 11q13 PRDX5 4.14 × 10⁻⁰⁷ 1.33 MS 12q13 Eos(IKZF4) 3.21 × 10⁻⁰⁸ 1.34 T1D, SLE ERBB3 1.27 × 10⁻⁰⁷ 1.34 T1D, SLE 6p21.32 MICA 1.19 × 10⁻⁰⁷ 1.44 ● T1D, RA, CeD, (HLA) UC, PS, SLE NOTCH41.03 × 10⁻⁰⁸ 1.61 T1D, RA, MS C6orf10 1.45 × 10⁻¹⁶ 2.36 ○● T1D, RA, PS,GV BTNL2 2.11 × 10⁻²⁶ 2.70 ○ T1D, RA, UC, CD, SLE, MS, GV HLA-DRA 2.93 ×10⁻³¹ 2.62 ○ T1D, RA, CeD, MS, GV HLA-DQA1 3.60 × 10⁻¹⁷ 2.15 ○● T1D, RA,CeD, MS, SLE, PS, CD, UC, GD, GV HLA-DQA2 1.38 × 10⁻³⁵ 5.43 T1D, RAHLA-DQB2 1.73 × 10⁻¹³ 1.60 RA HLA-DOB 2.07 × 10⁻⁰⁸ 1.72 Type I diabetes(T1D), rheumatoid arthritis (RA), celiac disease (CeD), multiplesclerosis (MS), system lupus erythematosus (SLE), Graves disease (GD),psoriasis (PS), Generalized Vitiligo (GV)

TABLE 7 Common Cause Model of Other Autoimmune Diseases. See also Jin etal., 2010, NEJM, 362:1686-1697. Stongest association Maximum Involved inother Region Gene (pvalue) odds ratio autoimmune disease  2q33.2 CTLA43.55 × 10⁻¹³ 1.44 T1D, RA, CeD, MS, SLE, GD ICOS 4.33 × 10⁻⁰⁸ 1.32 T1D,RA, CeD, MS, SLE, GD  4q27 IL21/IL2 4.27 × 10⁻⁰⁸ 1.34 T1D, RA, CeD, PS 6q25.1 ULBP6 4.49 × 10⁻¹⁹ 1.65 none ULBP3 4.43 × 10⁻¹⁷ 1.52 none 9q31.1 STX17 3.60 × 10⁻⁰⁷ 1.33 none 10p15.1 IL2RA 1.74 × 10⁻¹² 1.41T1D, MS, GD, GV (CD25) 11q13 PRDX5 4.14 × 10⁻⁰⁷ 1.33 MS 12q13 Eos(IKZF4) 3.21 × 10⁻⁰⁸ 1.34 T1D, SLE ERBB3 1.27 × 10⁻⁰⁷ 1.34 T1D, SLE 6p21.32 MICA 1.19 × 10⁻⁰⁷ 1.44 T1D, RA, CeD, UC, (HLA) PS, SLE NOTCH41.03 × 10⁻⁰⁸ 1.61 T1D, RA, MS C6orf10 1.45 × 10⁻¹⁶ 2.36 T1D, RA, PS, GVBTNL2 2.11 × 10⁻²⁶ 2.70 T1D, RA, UC, CD, SLE, MS, GV HLA-DRA 2.93 ×10⁻³¹ 2.62 T1D, RA, CeD, MS, GV HLA-DQA1 3.60 × 10⁻¹⁷ 2.15 T1D, RA, CeD,MS, SLE, PS, CD, UC, GD, GV HLA-DQA2 1.38 × 10⁻³⁵ 5.43 T1D, RA HLA-DQB21.73 × 10⁻¹³ 1.60 RA HLA-DOB 2.07 × 10⁻⁰⁸ 1.72 Type I diabetes (T1D),rheumatoid arthritis (RA), celiac disease (CeD), multiple sclerosis(MS), system lupus erythematosus (SLE), Graves disease (GD), psoriasis(PS), Generalized Vitiligo (GV).

TABLE 8 List of Genes implicated in AA. Black circles indicate genesthat affect NKG2D; white circles indicate genes that affect T-cellregulation; black circles with star indicate genes that affect endorgans Maxi- Stongest mum association odds Region Gene Function (pvalue)ratio  2q33.2 ○ CTLA4 T-cell proliferation 3.55 × 10⁻¹³ 1.44 ICOS T-cellproliferation 4.33 × 10⁻⁰⁸ 1.32  4q27 ○ IL21/IL2 T-, B- and NK-cell 4.27× 10⁻⁰⁸ 1.34 proliferation  6q25.1 ● ULBP6 NKG2D activatign 4.49 × 10⁻¹⁹1.65 ligand ● ULBP3 NKG2D activating 4.43 × 10⁻¹⁷ 1.52 ligand  9q31.1 ●STX17 premature hair 3.60 × 10⁻⁰⁷ 1.33 graying 10p15.1 ○ IL2RA T-cellproliferation 1.74 × 10⁻¹² 1.41 (CD25) 11q13 ● PRDX5 antioxidant enzyme4.14 × 10⁻⁰⁷ 1.33 12q13 ○ Eos T-cell proliferation 3.21 × 10⁻⁰⁸ 1.34(IKZF4) ERBB3 epidermal growth 1.27 × 10⁻⁰⁷ 1.34 factor receptor 6p21.32 ● MICA NKG2D activating 1.19 × 10⁻⁰⁷ 1.44 (HLA) ligand NOTCH4T-cell differentiation 1.03 × 10⁻⁰⁸ 1.61 C6orf10 1.45 × 10⁻¹⁶ 2.36 BTNL2T-cell proliferation 2.11 × 10⁻²⁶ 2.70 HLA-DRA Antigen presentation 2.93× 10⁻³¹ 2.62 HLA-DQA1 Antigen presentation 3.60 × 10⁻¹⁷ 2.15 HLA-DQA2Antigen presentation 1.38 × 10⁻³⁵ 5.43 HLA-DQB2 Antigen presentation1.73 × 10⁻¹³ 1.60 HLA-DOB Antigen presentation 2.07 × 10⁻⁰⁸ 1.72

Common pathways with other autoimmune diseases, including NK ligands inthe target organs include the following:

-   -   In RA, synoviocytes aberrantly express MIC ligands, leading to        autoreactive T cell stimulation.    -   In Celiac disease, MIC ligands are overexpressed in the gut        epithelium during active disease.    -   In Type 1 diabetes, Islet cells in prediabetic NOD mice express        RAE-1 ligand.    -   The aberrant expression of NK activating ligands in genetically        predisposed individuals can induce or exacerbate disease.

Candidate pathways in AA that present new opportunities for therapyinclude the following:

-   -   Members of the costimulatory receptor family, which have        complementary effects on T cell activation, and their balance        controls the overall outcome of immune and autoimmune responses.    -   Genes which enhance the NK cell cytotoxic response; triggers T        cells to produce proinflammatory cytokines; and induces CD4(+) T        cells to differentiate into Th17 cells.    -   Genes which pivotal role in regulating the adaptive immune        system by controlling the survival and proliferation of        regulatory T (Treg) cells, which are required for the        maintenance of immune tolerance.    -   Activating receptors expressed on natural killer (NK) cells,        NK1.1 (+) T cells, and T cells. NKG2D ligands include MHC class        I chain-related (MIC)A, MICB.

Candidate compounds for treatment of AA include compounds directed toJak/Stat signaling are described in Ivanenkov et al., Mini Rev Med Chem.2011 January; 11(1):55-78. which is hereby incorporated by reference inits entirety.

Example 2

The following genetic studies will be performed:

-   -   A replication study with another 1000 AA patients    -   Deep sequencing of candidate regions    -   Collection of 10,000 patients    -   Common Genetic Studies T1D, CeO and AA    -   An immunology study using NK activating ligands will be        performed using ULBP3, ULBP6—inducible transgenics, new mouse        models.

TABLE 9 Genes with Nominal Significance on the GWAS studies Count Min p-Min p- of SNPs value value Gene Mb <1 × 10⁻⁴ observed imputed AutoimmuneReports GO classification Chromosome 2 HDAC4 240.03 1 8.10E−05 5.59E−05inflammatory response Chromosome 3 CACNA2D3 55.02 1 7.28E−05 1.47E−05CeD Chromosome 5 IL13 132.02 2 1.87E−06 Asthma immune responseChromosome 6 HLA-G 29.94 1 1.07E−04 5.54E−06 RA, MS, SLE, PS, T1D,Asthma immune response HLA-A 30.01 1 1.00E−04 2.72E−05 MS, T1D, PS, GD,Asthma, Vitilago immune response MICB 31.59 2 1.89E−05 1.97E−05 MS, T1D,UC, RA, CeD, Asthma immune response TAP2 32.91 1 6.42E−06 1.28E−05 T1D,RA, SLE, PS, GD immune response Chromosome 7 IL6 22.72 2 7.72E−054.84E−05 RA, T1D, CeD inflammatory response CHCHD3 132.44 1 3.07E−052.02E−05 CeD Chromosome 8 CSMD1 3.02 1 8.65E−05 8.38E−05 CeD, MS, PSChromosome 12 IFNG 66.84 1 1.55E−05 1.29E−05 CeD, T1D, RA, MS, SLE, PS,GD, Asthma IL26 66.87 2 7.18E−05 6.45E−05 MS, Asthma immune repsonseChromosome 16 KIAA0350 11.11 6 1.77E−05 1.15E−05 T1D, MS, ThyroidDisease (CLEC16A) SOCS1 11.24 2 1.16E−05 8.66E−06 CeD, T1D, AsthmaChromosome 18 ANKRD12 9.25 2 3.59E−05 1.55E−05 PTPN2 12.80 1 4.09E−063.38E−07 CD, T1D

TABLE 10 Pathway analysis of nominal genes Category Term Genes hsa05332Graft-versus-host disease IL6, IFNG, HLA-A, HLA- DMA, HLA-G hsa04612Antigen processing and HSPA1L, TAP2, HLA-A, presentation HLA-DMA, HLA-Ghsa04630 Jak-STAT signaling pathway SPRY2, IL6, SOCS1, IFNG, IL26, IL13hsa05330 Allograft rejection IFNG, HLA-A, HLA-DMA, HLA-G hsa04940 Type Idiabetes mellitus IFNG, HLA-A, HLA-DMA, HLA-G hsa04650 Natural killercell mediated MICB, IFNG, HLA-A, cytotoxicity HLA-G hsa05320 Autoimmunethyroid disease HLA-A, HLA-DMA; HLA-G hsa05416 Viral myocarditis HLA-A,HLA-DMA, HLA-G hsa04060 Cytokine-cytokine receptor IL6, IFNG, IL26, IL13interaction

Example 3—Study Samples, Genotyping, Quality Control and PopulationStratification

Alopecia areata (AA) is a major medical problem and is the mostprevalent autoimmune disease in the US (Table 11), affectingapproximately 4.6 million people, including males and females across allethnic groups, with a lifetime risk of 1.7% (Kyriakis K P, PaltatzidouK, Kosma E, Sofouri E, Tadros A, Rachioti E. Alopecia areata prevalenceby gender and age. J Eur Acad Dermatol Venereol. 23:572-3, 2009).Additionally, AA represents the second most common form of human hairloss, second only to androgenetic alopecia, and causes significantdisfigurement and psychological distress to affected individuals (FIG.1). AA affects more individuals than most other autoimmune diseasescombined, including lupus erythematosus (LE), type 1 diabetes (T1D),psoriasis, multiple sclerosis (MS) and rheumatoid arthritis (RA) (Table11).

TABLE 11 Automimmune Disease Prevalence Autoimmune Disease Rate per100,000 Alopecia Areata 1700 Psoriasis  696-1527 Rheumatoid arthritis310-800 Type 1 diabetes 227-355 Multiple sclerosis 177-358 Systemiclupus erythematosus  34-150

In stark contrast to these other conditions, research into thepathogenesis and the development of innovative therapies in AA haslagged far behind. This can be due in part to the perception that AA ismerely a cosmetic disorder. In reality, AA carries one of the highestburdens among any skin diseases, particularly among children andadolescents whose self-image is so closely linked to their appearance(Bickers D R, Lim H W, Margolis D, Weinstock M A, Goodman C, Faulkner E,Gould C, Gemmen E, Dall T; American Academy of Dermatology Association;Society for Investigative Dermatology. The burden of skin diseases: 2004a joint project of the American Academy of Dermatology Association andthe Society for Investigative Dermatology. J Am Acad Dermatol. 55:490-5,2006).

Despite its high prevalence, there are no evidence-based treatments forAA. A comprehensive Cochrane analysis assessment of seventeen randomizedclinical trials (RCTs) involving a total of 540 participants found noproven treatment of AA (Delamere F M, Sladden M M, Dobbins H M,Leonardi-Bee J. Interventions for alopecia areata. Cochrane DatabaseSyst Rev. 2:CD004413, 2008). Each trial included from 6 to 85participants and assessed a range of interventions that included topicaland oral corticosteroids, topical cyclosporin, photodynamic therapy andtopical minoxidil. Overall, none of the interventions showed significanttreatment benefit in terms of hair growth when compared with placebo. Itwas concluded that, few, (if any), treatments for AA are proven to beeffective. No RCTs on the use of diphencyprone, dinitrochlorobenzene,intralesional corticosteroids or dithranol were found, although thesedrugs are commonly used for the treatment of AA. Similarly, althoughtopical corticosteroids and minoxidil are widely prescribed and appearto be safe, there is no convincing evidence that they are beneficial inthe long-term. Most trials have been poorly reported and/or are so smallthat any important clinical benefits are inconclusive. Theseobservations underscore the importance of defining the genetic basis ofAA and determining its pathogenesis, so that rational therapeuticapproaches can be developed and translational research can begin.

Pre-Clinical Evaluation of Jak/Stat Targeting in Alopecia Areata

The NKG2D receptor (NKG2D), functions to eliminate cells emitting dangersignals and dysregulation of this recognition process often leads todevelopment of autoimmunity. The influences of JAK and NF-kb inhibitionwill be pursued, where, without being bound by theory, NKG2DL expressionwill be inhibited at the transcriptional level. INCB 18424, a JAK1/JAK2inhibitor (po/topical), is in Phase II Clinical Trials forMyeloproliferative/Myelofibrosis Disorders and in Psoriasis.

TABLE 12 Small Molecule Inhibitors of Jak1/Jak2 Small moleculesJAK1/JAK2 PTKi: ATP-Binding Interferon Topical Phase Inhibitor CleftCompetitive Signaling II/Psoriasis (INCB18424) Antagonist

GWAS studies revealed a number of risk loci shared by with other formsof autoimmunity, such as rheumatoid arthritis (RA), type I diabetes(T1D), celiac disease (CeD), systemic lupus erythematosus (SLE),multiple sclerosis (MS) and psoriasis (PS), in particular, CTLA-4,IL2/IL2RA, IL21 and genes critical to Treg maintenance.

A genetic basis of AA provide avenues of exploration for therapies basedon the underlying mechanisms of AA. Such therapies will focus not onlyon T cell subsets and mechanisms common to other forms of autoimmunity,but also on unique mechanisms that involve signaling pathways ofJak1/Jak2 and downstream effectors.

The origin of autoimmunity in AA can reside in the hair follicle itself.The studies herein are focused on defining putative danger signals inthe hair follicle that contribute to the pathogenesis of AA. Pathogenicalleles that reside within the MHC, which can contribute to immunedysregulation driving the pathogenesis of AA, will also be identified.This can be due in part to the importance of antigen-presenting cells(APCs) in the presentation of danger signals as neoantigens to theimmune system.

The mechanisms of Jak1/Jak2 signaling will be investigated, and theimportance of Jak1/Jak2/Stat1/Stat2 interactions as drivers of diseaseinduction will be evaluated. Pharmacologic approaches interrupting theJak1/Jak2/Stat1/Stat2 signaling will be pursued.

C3H IFN-γ deficient mice are protected from AA development. It is shownthat IFNs are capable of upregulating NKG2DL in cultured human dermalkeratinocytes and fibroblasts. Further NKG2D-cross-linking induces IFN-γproduction by innate NK, NKT and γδ T cells, thus creating a potentialpositive feedback loop, driving adaptive autoreactive immunity. Thusadoptive transfer and antibody depletion/blockade experiments willdefine the NKG2D-bearing cells involved.

Pharmacologic interventions aimed at interruption of the CD8 NKG2D axiswill be examined. Interfering with the upregulation of NKG2DL or theactivation of NKG2D-bearing cells are both rational approaches that willbe pursued. It is shown that the IFN→JAK/STAT pathway can induce NKG2DLexpression.

Whether the IFN→JAK/STAT pathway is pivotal to NKG2DL induction innormal and diseased skin will be assessed. The IFN JAK/STAT signalingpathway can be effectively inhibited with topical JAK1/JAK2 inhibitors,including an agent that has had early clinical success in Psoriasis. Ifthe Type I interferon response underlies NKG2D ligand upregulation,JAK1/JAK2 inhibitors can block this induction, as well as block theIFN-gamma dependent components of the adaptive immune response.

REFERENCES

-   1. Safavi K H, Muller S A, Suman V J, Moshell A N, Melton L J, 3rd.    Incidence of alopecia areata in Olmsted County, Minn., 1975    through 1989. Mayo Clin Proc 1995; 70:628-33.-   2. Jelinek J E. Sudden whitening of the hair. Bull N Y Acad Med    1972; 48:1003-13.-   3. Ito T, Ito N, Saatoff M, et al. Maintenance of hair follicle    immune privilege is linked to prevention of N K cell attack. The    Journal of investigative dermatology 2008; 128:1196-206.-   4. Todes-Taylor N, Turner R, Wood G S, Stratte P T, Morhenn V B. T    cell subpopulations in alopecia areata. J Am Acad Dermatol 1984;    11:216-23.-   5. Gilhar A, Landau M, Assy B, et al. Transfer of alopecia areata in    the human scalp graft/Prkdc(scid) (SCID) mouse system is    characterized by a TH1 response. Clin Immunol 2003; 106:181-7.-   6. Gilhar A, Shalaginov R, Assy B, Serafimovich S, Kalish R S.    Alopecia areata is a T-lymphocyte mediated autoimmune disease:    lesional human T-lymphocytes transfer alopecia areata to human skin    grafts on SCID mice. J Investig Dermatol Symp Proc 1999; 4:207-10.-   7. Zoller M, McElwee K J, Engel P, Hoffmann R. Transient CD44    variant isoform expression and reduction in CD4(+)/CD25(+)    regulatory T cells in C3H/HeJ mice with alopecia areata. The Journal    of investigative dermatology 2002; 118:983-92.-   8. McElwee K J, Freyschmidt-Paul P, Hoffmann R, et al. Transfer of    CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(−)    cells promote systemic alopecia areata and CD4(+)/CD25(+) cells    blockade disease onset in the C3H/HeJ mouse model. The Journal of    investigative dermatology 2005; 124:947-57.-   9. Ito T, Meyer K C, Ito N, Paus R. Immune privilege and the skin.    Curr Dir Autoimmun 2008; 10:27-52.-   10. McDonagh A J, Tazi-Ahnini R. Epidemiology and genetics of    alopecia areata. Clin Exp Dermatol 2002; 27:405-9.-   11. van der Steen P, Traupe H, Happle R, Boezeman J, Strater R,    Hamm H. The genetic risk for alopecia areata in first degree    relatives of severely affected patients. An estimate. Acta Derm    Venereol 1992; 72:373-5.-   12. Jackow C, Puffer N, Hordinsky M, Nelson J, Tarrand J, Duvic M.    Alopecia areata and cytomegalovirus infection in twins: genes versus    environment? J Am Acad Dermatol 1998; 38:418-25.-   13. Martinez-Mir A, Zlotogorski A, Gordon D, et al. Genomewide scan    for linkage reveals evidence of several susceptibility loci for    alopecia areata. Am J Hum Genet 2007; 80:316-28.-   14. Nakamura et al., 2008, Controlled Delivery of T-box21 Small    Interfering RNA Ameliorates Autoimmune Alopecia (Alopecia Areata) in    a C3H/HeJ Mouse Model, Am J Pathol., 172(3): 650-658.-   15. Freyschmidt-Paul, K. J. McElwee, R. Hoffmannl, J. P.    Sundberg, M. Vitacolonna, S. Kissling, M. Zoller, 2006,    Interferon-gamma-deficient mice are resistant to the development of    alopecia areata, Br J Dermatol., 155(3):515-21.-   16. Gilhar A., Kam Y., Assy B. and Kalish R., 2005, Alopecia areata    induced in C3H/HeJ mice by interferon-gamma: evidence for loss of    immune privilege, Journal Invest. Dermatol., 124(1):288-9.-   17. Wong et al., 2005, Inhibitors of the tyrosine kinase signaling    cascade for asthma, Curr. Opin. Pharmacol., 5(3):264-71.-   18. Mourich and Iverson, 2009, Splicing in the immune system:    Potential targets for therapeutic intervention by antisense-mediated    alternative splicing, Curr. Opinion Mol. Ther., 11(2): 124-132.-   19. Jin et al., 2010, Variant of TYR and Autoimmunity Susceptibility    Loci in Generalized Vitiligo, NEJM, 362:1686-1697.

Example 4

Specific autoimmune mechanisms underlying Alopecia Areata (AA) haveremained obscure and therefore clinical investigation of AA hashistorically lagged behind other autoimmune diseases. Despite the largenumber of new biotherapeutics available for clinical evaluation inautoimmunity, intralesional or systemic steroids remain the standard ofcare and only two small randomized clinical trials have been publishedin the last decade. The insights provided by the discovery of GWAS genesprovide a roadmap for closing this gap, by investigating GWAS-identifiedimmunological pathways both for mechanistic and therapeutic relevance.

Identify Effective, Clinically-Relevant, Therapies in Mouse Models ofAlopecia Areata.

In the normal state, hair follicles (HF) represent an immuneprivileged(IP) sanctuary, expressing little or no classical HLA class I molecules.In active AA, the IP status is lost, since HLA class I expression andNKG2D ligand expression are markedly upregulated. Given the stronggenetic association of NKG2D ligand loci with human AA, without beingbound by theory, aberrant NKG2DL upregulation and persistent NKG2Dactivation on immune effector cells drives inflammatory AA initiationand progression. Without being bound by theory, in AA, as for CeliacDisease, IL-IS can drive CD8 T cell effector differentiation enablingpromiscuous “NK-type” CD8 cytotoxicity. Indeed, the local HFIL-15/NKG2DL inflammatory signals are accompanied by a dense infiltrateof CD8⁺ T cells expressing NK markers, likely the critical AA immuneeffectors responsible for IFNγ production and HF cytotoxicity. Thesecollective observations, made in both the human and mouse, invitetherapeutic evaluation.

The therapeutic potential of blocking NK-like reprogramming andcytotoxicity by interfering with relevant cytokines (IFN-γ□ and relevantimmunoreceptor signaling pathways (NKG2D). A graft model of alopecia inwhich all grafted C3H mice develop AA in a timely manner, within 12weeks, will be used. This greatly accelerates drug screening sinceevaluation of prevention/reversal of spontaneous disease in C3H/HeJ isotherwise impractical given the late onset (>6 months of age) and lowcumulative incidence (15%). Both small molecule approaches, that havethe advantage of potential topical delivery, and biotherapeutic proteins(antibodies) that have superior biologic specificity, will be evaluated.The overarching goals are to use biotherapeutics to establish criticalimmune pathways that might be targeted by more clinically tenable, smallmolecule topical approaches.

Identify Disease-Associated AA Biomarkers and their Reversal withEffective Therapy in C3H Mice.

Approaches using GWAS and transcriptional profiling, for example, aswell as approaches using immunostains and FACs of T cell subsets, forexample, have been taken to identify several pathogenic AA biomarkers inthe skin and blood of AA mice. These mouse studies will continue toinform developing a translational biomarker platform in human AA. In theskin, the studies in alopecic C3H/Hej mice demonstrated that IFN-inducedgenes are dramatically upregulated, including the chemokines CXCL9-11,which are likely responsible for recruitment of a dense infiltrate ofHF-associated IFN-γ-producing CXCR3⁺NKG2D-bearing CD8 T effectors.Spectratype data provide striking evidence that these circulating NKG2D⁺CD8 T effectors are bona fide autoantigen-driven effector T cells thatincrease with disease progression. Temporal assessment of theseserologic, cutaneous and cellular biomarkers in mice during treatmentwill enable one to identify dynamic, mechanistically important,prescient inflammatory biomarkers that predict therapeutic outcome andinform clinical biomarker discovery/utilization.

Preclinical Validation of Targeted Approaches Using AA Human Tissues ExVivo.

To expedite clinical development of treatments that demonstrate“proof-of-concept” in the AA mouse model, human AA tissues will be usedfor target validation and correlative studies. The “crawl-outs” systemwill provide a convenient ex vivo human bioassay to validate that agentsthat reverse mouse AA translate to the human, effectively inhibitinghuman AA T cell survival/expansion/function.

Just as the human DNA from thousands of alopecic subjects provided GWASgenes and unique insight into the human disease, human tissues, bloodand skin from AA subjects will be used to continue to interrogate thesegenetically implicated immune pathways ultimately for therapeuticutility. Conceptually, the mouse model of alopecia areata this humanpursuit, providing the pre-clinical testing grounds for therapeuticapproaches in alopecia areata.

Autoimmune diseases affect an estimated 23 million individuals in the US(1) and although the prevalence of AA is not established, it is one ofthe most prevalent autoimmune diseases (2, 3). In 1999-2000, there werean estimated 2.4 million office visits for AA, half of which by patientsin their 20s and 30s. AA causes significant disfigurement andpsychological distress to affected individuals and carries one of thehighest burdens among any skin diseases, particularly among children andadolescents whose self-image is so closely linked to their appearance(4). At present, the prognosis of AA is unpredictable and there is nodefinitive treatment. Current therapies include steroids and topicalimmune therapy, and induce durable remissions in only one third ofpatients (5, 6). Despite its high prevalence, a comprehensive Cochraneanalysis assessment concluded that there are no evidence-basedtreatments for AA (7).

In stark contrast to other autoimmune conditions, research into thepathogenesis and the development of innovative therapies in AA haslagged far behind. In the era of targeted immunological therapies, onlytwo clinical studies evaluating protein biotherapeutics in AA have beenreported (8, 9) (both targeting T cell trafficking). In part this hasbeen due to the limited understanding of specific AA immune pathwaysthat would prompt evaluation of rational therapeutic approaches. Basedon new understanding of the genetic and pathogenic basis of AApre-clinical proof-of-concept data that will prompt clinical evaluationof rational therapeutic approaches in this disease will be obtained.

The GWAS (10) study revealed a number of risk loci shared by with otherforms of autoimmunity, such as RA, T1D, celiac disease (CeD), SLE, MSand PS, in particular, CTLA4, IL2/IL2RA, IL21, NKG2D Ligands and genescritical to Treg function (Eos). The genetic commonality with RA, T1D,and CeD is especially noteworthy in light of the pathogenic significanceof the expression of an NK ligand in the end organ (synovium, islet, gutand skin), and the involvement of the NKG2DL/NKG2D pathway in thepathogenesis of each of these three diseases (11, 14). One advantage ofimmunotherapeutic studies in the skin is the relative ease of access ofthe target organ. Thus, the studies herein examining the skin canprovide important insight into NKG2D and IL-15 triggered CD8cytotoxicity, IFN-triggered injury, etc., that impact the understandingof these other related human diseases in which the target organ is notaccessible. Indeed positive studies in any one of these autoimmunediseases that share a common cause can serve as the basis for commontreatments.

Specific autoimmune mechanisms underlying AA have remained obscurebeyond the prevailing view that it is the result of T cell mediatedattack of the hair follicle. In the normal state, hair folliclesrepresent an immuneprivileged (IP) sanctuary (15-17), expressing littleor no classical HLA class I molecules and instead express the inhibitoryHLA ligands HLA-E and HLA-G, thus simultaneously avoiding immunerecognition by class I restricted CD8 cells while negatively regulatingNK cells, that might otherwise react to “missing self” (16, 18-21). Inactive AA, the IP status of the normal hair follicle is not operative,since HLA class I expression is markedly upregulated. Previous studieshave noted both CD4 and CD8 T cell infiltrates surround the hairfollicle in alopecic areas of skin in the mouse and human (22, 23).Indeed transfer of total T cells, but not B cells or sera, can transferdisease from an alopecic mouse or human to a normal WT (24) or SCIDmouse (25), implicating effector T cells as the pathogenic cells.However molecular mediators of site-specific inflammation have not beenpreviously identified. Guided by the GWAS findings and previousimmunobiological studies, new insight into this common autoimmunedisorder is provided, that will lead to the development of innovative,rationally targeted treatments to fill the unmet needs of patients withAA.

AA Immunological Pathways Identified by GWAS Studies

The most highly-significant genetic loci found associated with alopeciaareata are shown in Table 13. These seminal studies have been validatedin a confirmatory study in a distinct population. Underlined genes areHF expressed genes while bold genes are genes expressed in immune cells.Overall, many susceptible loci are shared by other tissue-specificautoimmune states, with Type I diabetes exhibiting the most strikingoverlap (right column). The commonality with RA, T1D and CeD isparticularly noteworthy, as NKG2D has been shown to have a significantrole in the pathogenesis of each of these three diseases (11-14). Thusinsight in any one of these disease/model systems is likely to shedlight on common pathogenic pathways shared by multiple autoimmunestates. The most highly significant alleles identified (other than HLA)were four NKG2D ligands (UBLP-3/UBLP-6/MICA/MICB in Bold italics) andone inhibitory ligand (HLA-G). The NKG2D receptor (NKG2D), functions toeliminate cells emitting danger signals and dysregulation of thisrecognition process often leads to development of autoimmunity (26).Other HF expressed genes that contribute to risk include HLA, antigenprocessing genes (TAP) and PRDX/STX17.

TABLE 13 Genetic loci associated with AA.  2q33.2 CTLA4 Inhibits costim3.6 × 10⁻¹³ T1D, RA, CeD, MS, SLE, GD ICOS T-cell costim 4.3 × −10⁻⁰⁸T1D, RA, CeD, MS, SLE  4q27 IL21 NK function/Th17 4.3 × 10⁻⁰⁸ T1D, RA,CeD, PS IL2 T-cell./Treg prolif 3.0 × 10⁻⁰⁴ T1D, RA, CD, MS, CD, SLE,PS, GD  6q25.1 ULBP6 NKG2D ligand 4.5 × 10⁻¹⁹ None ULBP3 NKG2D ligand4.4 × 10⁻¹⁷ None  9q31.1 STX17 hair graying 3.6 × 10⁻⁰⁷ None 10p15 IL2RAT-cell prolif./Treg 1.7 × 10⁻¹² T1D, MS, GD 11q13 PRDX5 antiox enz 4.1 ×10⁻⁰⁷ MS 12q13 Eos T-reg 3.2 × 10⁻⁰⁸ T1D, SLE programming  6p21.3 MICANKG2D ligand 1.2 × 10⁻⁰⁷ T1D, RA, CeD, UC, PS, SLE (HLA) NOTCH4Hematopoiesis 1.0 × 10⁻⁰⁸ T1D, RA, MS BTNL2 T cell costim 2.1 × 10⁻²⁶T1D, RA, UC, CD, SLE, MS HLA-DRA Ag presentation 2.9 × 10⁻³¹ T1D, RA,CeD, MS HLA-DQA1 Ag presentation 3.6 × 10⁻¹⁷ T1D, RA, CeD, MS, SLE, PS,CD UC, GD HLA-DQA2 Ag presentation 1.4 × 10⁻³⁵ T1D, RA HLA-DQB2 Agpresentation 1.7 × 10⁻¹³ RA HLA-DOB Ag presentation 2.1 × 10⁻⁰⁸ SLE 5IL-13 Th2 polarization 1.9 × 10⁻⁰⁶ Asthma 6 HLA-G NKG2A inhibition 1.0 ×10⁻⁰⁴ RA, MS, SLE, PS, T1D, Asthma HLA-A Ag presentation 1.0 × 10⁻⁰⁴ MS,T1D, PS, GD, Asthma, Vitiligo MICB NKG2D ligand 1.9 × 10⁻⁰⁵ MS, T1D, UC,RA, CeD, Asthma TAP2 Ag presentation 6.4 × 10⁻⁰⁶ T1D, RA, SLE, PS, GD 7IL6 Inflammation 7.7 × 10⁰⁵ RA, T1D, CeD 12 IFNG Th1/inflamm 1.5 × 10⁰⁵CD, T1D, RA, MS, SLE, PS, GD, Asthma IL26 T polarization 7.2 × 10⁻⁰⁵ MS,Asthma 16 CLEC16A C-type lectin 1.8 × 10⁻⁰⁵ T1D, MS, GD receptor SOCS1JAK/STAT inh 1.2 × 10⁻⁰⁵ CeD, T1D, Asthma 18 PTPN2 phosphatase 4.2 ×10⁻⁰⁶ CeD, T1D

IFN-γ production by activated NKG2D-bearing CD8 effectors can reverseimmunologic privilege and perpetuate the inflammatory loop.

IFN-γ related pathways can be undermined with existing therapeuticsunder clinical investigation, expediting the path to translation. Overthe last ten years, several small molecule inhibitors of proteintyrosine kinases (PTKi) have been successfully developed clinically bothfor oral and topical delivery. PTKi approaches will be pursued toblocking CD8 mediated inflammatory responses in AA, namely inhibition ofJAK/STAT signaling, responsible for cytokine responsiveness, downstreamof the CD8 effector cytokine IFN-γ.

Identify Clinically-Relevant Therapies Targeting Effector T CellResponses in Alopecic Mice.

Using immunostaining and flow cytometric analysis of total cellularpopulations from AA skin, CD8⁺NKG2D⁺ T cells were found infiltrating thehair follicle (FIG. 17) and expanded in AA but not normal skin (FIG. 18and FIG. 4A). Furthermore AA mice exhibit cutaneous lymphadenopathy, atleast in part due to dramatic systemic expansion of the CD8⁺NKG2D⁺population, in both the AA cutaneous lymph node and in the blood,representing fully 6+/−1% of total LN cells are 2.4+/−1.3% of totalPBMCs, each more than ten-fold higher than that seen in normal C3H mice.These CD8⁺NKG2D⁺ T cells are potently cytotoxic against HF dermal sheathcells (FIG. 21). Further immunophenotypic analysis of CD8⁺NKG2D⁺ T cellsrevealed that this population expressed the T memory marker CD44⁺ andmultiple “NK markers”, including DX5, NKG2A/C/E, and NKp46 (FIG. 19).

To provide proof that the circulating CD8⁺NKG2D⁺ population representedthe bona fide “AA-specific” effector population, without being bound bytheory, the CD8⁺NKG2D⁺ T cells found in the lymph node can express asimilar TCR repertoire to the total T cells found infiltrating AA skin.FIG. 20 shows that CD8⁺NKG2D⁻ lymph node populations exhibit a normaldistribution of CDR lengths, a pattern reflective of an unrestrictedpolyclonal T cell population. In contrast, the TCR repertoire of skin Tcells are highly restricted with nearly all Vβ family members dominatedby a small number of CDR lengths, indicative of an oligoclonalpopulation of antigen-driven T cells. CD8⁺NKG2D⁺ T cells from thecutaneous lymph node cells are also highly restricted and show astrikingly similar pattern with total cutaneous T cells.

These data confirm that CD8⁺NKG2D⁺ T cells contain most of theoligoclonal T cell populations found in the skin. Moreover the evidencefor the expansion of common oligoclonal T cells in the skin and lymphnode argues against the possibility that these LN populations (or evenmarkers) are expanded/activated as a reactive process to inflammatoryevents. For instance, Treg cells (CD4⁺CD25⁺FoxP3⁺) are also increased innumbers in AA lymph nodes, however, this is likely a reactive process,since their spectratype is diverse and unlike those present in the skin.Note that some TCR/clones are found in skin but not in drainingcutaneous lymph nodes, e.g., V1310. These likely include CD4⁺ T cellpopulations found in AA skin but not present in the sorted CD8+LNfraction. These data establish that the CD8⁺NKG2D⁺ T cells are thepathogenic NKG2D⁺ effectors in mouse AA, establishing a parallel of themouse model to human AA.

Pre-Clinical AA Model to Establish Proof of Concept for TherapeuticApproaches In Vivo

C3H/HeJ Skin Graft Model:

The C3H/HeJ mouse model for alopecia areata has been studied since itwas first reported this model in 1994 (29, 30, 31). While other mousemodels for alopecia areata were found (32), this model remains the moststudied and used in the field today. Having done a variety of drugtrials with this model over the years (33), a variety of protocols havebeen optimized (34).

Full thickness skin grafting from alopecic C3H/HeJ mice to 2-3 month-oldunaffected C3H/HeJ female mice results in patchy alopecia within 10weeks of grafting in all recipients, progressing to generalized alopeciaat 20 weeks. A variety of comprehensive and efficient histologicalscoring systems are employed building on the traditional training ofhistopathologists where the adjective used in descriptions (normal, 0;mild, 1; moderate, 2; severe, 3; and extreme, 4) are converted to anumerical value for importation into a relational database (MouseDisease Information System [or MoDIS]:research.jax.org/faculty/sundberg/registration.php) (35, 36). Thisdatabase is exportable via Excel for importation into a statisticalanalysis or other software program. Alopecia areata is graded based onseverity, location of inflammatory cells, types and relative numbers ofinflammatory cells (granulocytes, mast cells, lymphocytes), folliculardystrophy, etc. Molecular RNA and protein biomarkers will beincorporated in this panel, and generation and analysis of this multiplevariable database will be done.

Protocol#1:

In the prevention setting, recipient mice will be treated beginning thefirst week after grafting.

Protocol#2:

For treatment, grafted mice with early-established AA will be treated toreverse AA progression.

Agents will first be examined in the prevention model. Agents thatsuccessfully prevent AA will then move forward for secondary evaluationin the setting of established disease, while those that fail the firsttest will be discarded, permitting entry of other innovative approaches.The following are the initial therapeutic approaches, both specificbiologics and small molecules, chosen for preclinical evaluation.

Small molecule JAK protein tryosine kinase inhibitors (PTKis) thattarget activation pathways involved in the T effector response will betested. These PTKis are already in Phase III clinical trials easingtranslation and potential clinical development of “NK-type” Tcell-targeted topicals.

Therapeutic Interventions with Small Molecule PTKi Interventions

Topical delivery has obvious advantages in limiting systemic exposuresand toxicities. Pre-clinical and clinical studies have demonstrated thatsmall molecule PTKi delivery in cream can overcome the skin barrier andachieve effective local concentrations in the dermis with limitedsystemic exposure (78). Key PTKs pivotal to effector T responses in AAwill be targeted, namely the cytokine IFNγ.

Targeting IFNs with Jak1/2 Inhibitors:

Interferons are an attractive therapeutic target in AA since they likelyparticipate at several steps in the inflammatory response; includingeliminating HF immunologic privilege and inducing the cellularinflammatory response. Interferons upegulate several relevantpro-inflammatory molecules in the hair follicle end organ, includingNKG2DL, adhesion molecules (e.g., ICAM-1), antigenicprocessing/presentation (TAP 1/2, LMP, proteosome, MHCI and II) andmoreover drive the immune effector response (increased Th1-typeresponses, DC activation and IFN-γ-mediated cytotoxicity). In theC3H-HeJ mouse model of AA, IFN-γ is required for pathogenesis (89, 90)and administration of interferon-γ accelerates disease (91).Importantly, administration of IFN-γ neutralizing antibodies reverses AApathogenesis in the C3H-HeJ mouse (92, 93). Likewise, in the human,alopecia areata has been noted in several series as a side-effect ofType I interferon therapy (94-104) and transcriptional profiles of humanAA skin have noted a Type I IFN response in lesional biopsies (105) andTh1 skewing and elevated IFN-response cytokines/chemokines in theperipheral blood (FIGS. 22, 23) (106-110) and reviewed in (111). T cellsobtained from AA skin biopsy explants were uniformly CD8⁺NKG2D⁺ cellsand when stimulated produced IFN-γ (FIG. 21A) but not L-4 or IL-17. Instark contrast, the majority of T cells obtained from normal skinexplants are CD4⁺ (112, 113). The situation is similar in AA mice, IFN-γproducing CD8 T cells are expanded in the cutaneous lymph node (FIGS.19, 21). In the skin, our transcriptional profiling studies of totalskin isolated from lesional vs. non-lesional C3H skin has identified aninterferon response as the dominant signature with 17 of the top 20upregulated genes being interferon response genes (FIG. 22). Indeed,IFNs upregulate NKG2DL expression on hair follicle targets, closing anIFN-mediated pathogenic circle, in which IFN-γ made by CD8 effectorsdrive the HF NKG2D ligands that lead to CD8⁺NKG2D⁺ activation.

Clinical JAK1/2 Inhibitors:

Both blocking antibodies (114, 115) and small molecule PTKis are underclinical development targeting the IFN response in autoimmunity. IFNinduced STAT1/2 activation is mediated by Jak1/Jak2/Tyk kinases andclinical data have emerged using oral Jak1/Jak2 PTKis in severalautoimmune conditions, including RA and psoriasis. Topical therapieshave the added advantage of an improved therapeutic index limiting thepotential for systemic immunosuppression. INCB018424 inhibits Jak1 andJak2 (IC₅₀=1 nM) and is the only topical JAK inhibitor (78) currently inclinical investigation and its safety profile to date has beenacceptable. Proof-of-concept with oral delivery has been demonstrated ina Jak-dependent disease (myelofibrosis) (116) and in a dermatologicaldisease driven by local cytokine production (psoriasis models) (78). Inpsoriasis subjects, topical INCB018424 induced rapid clinical responsesand normalization of cutaneous Th1/Th17 cytokine responses (unpublisheddata from clinicaltrials.gov/ct2/show/NCT00820950). Taken together,topical INCB018424 is a safe intervention predicted to have efficacy inAlopecia Areata, an inflammatory state dominated by an IFN-signature.

Approach: Protocol #1: Systemic Delivery of INCB018424:

Twice daily (90 mg/kg) orogastric gavage provides good systemic exposuregiven the t_(1/2) half-life of 3-6 hours in the rodent. In this mannerwe can assess if JAK1/2 inhibition prevents AA progression.

Protocol #2: Topical Delivery of INCB018424 (1.5%):

Reversal of patchy AA by topical JAK1/JAK2 inhibitor. Daily topicaltreatment (78) will begin to new affected patchy areas in grafted micethat begin to emerge 6-10 weeks after grafting. Topical treatment can berestricted to specific areas of mouse skin by appropriate bandaging toprevent systemic exposures from grooming, licking etc. Importantly, bytreating affected areas we can assess the clinically relevant questionof whether topical exposure can reverse AA progression.

Identification of Murine Biomarkers of AA Disease Activity that InformClinical Investigation

The identification of immunological events that accompany treatmentoutcome in these pre-clinical studies, is important, both to validatethe proposed mechanism of action and to provide useful biomarkers thatwill inform potential clinical evaluation of these same therapies in AApatients. Since in clinical evaluation, biospecimens will be limited toblood and skin samples, the focus will be on mouse biomarkers in themouse that are easily sampled from these sites. Approaches using GWASand transcriptional profiling, for example, as well as approaches usingimmunostains and FACs of T cell subsets, for example, have been taken toidentify several pathogenic biomarkers in the skin and blood of AA mice.The studies in alopecic C3H/HeJ mice have already identified severalskin biomarkers upregulated in AA, such as IFN-induced genes (FIG. 22).Spectratype data (FIG. 20) has provided striking evidence that thesecirculating NKG2D⁺ CD8 T effectors are bona fide autoantigen-driveneffector T cells. Temporal assessment of these serologic, cutaneous andcellular biomarkers in mice, with and without treatment, will enable oneto identify dynamic, mechanistically important, prescient inflammatorybiomarkers that predict therapeutic outcome and inform clinicalbiomarker discovery/utilization.

Cellular Biomarkers of Circulating Immune Effectors in AA:

“NK-type” CD8 T cells are expanded in the skin, blood and lymph node inAA mice and are a valuable tool to investigate pathogenesis. Moreover,the cellular subset appears directly relevant to the human disease.

Spectratype-Based Imunomonitoring:

NK-type CD8 T cells will be identified in AA mice in the circulation byflow cytometry of 50 μl of whole blood (FIG. 18). The total numbers andspectratype of this CD8⁺NKG2D⁺ sorted T cell subset, will be assessedevery three weeks after grafting to monitor the evolution of clonaldominance/epitope spreading within a single mouse. Spectratype analysiswill be used to confirm that immunophenotypic loss of CD8⁺NKG2D⁺ T cellsis due to treatment-related elimination of the circulating alopecic Tcell clones. This non-invasive immunomonitoring approach will be used tofollow mice on all treatments. For instance, 3 weeks of treatmenttreatment with the JAK1/2 inhibitor INCB018424 did not. Without beingbound by theory, JAK1/2 inhibitors are expected to abrogate thedownstream inflammatory consequences of T cell derived effectorcytokines (IFN-γ, IL-17) in the target tissue.

Immune “Proteomic” Signatures in Serum:

Cytokines and chemokines in the serum are a reflection of theinflammatory milieu in the target organ. The data in the humandemonstrate elevations of several cytokines and chemokine some of whichcorrelate with disease activity (for instance IL-8), extending theobservations of prior studies (106, 107, 109). Consistent with ouroverall findings implicating IL-15 and IFN-γ in AA pathogenesis, it isseen that IFN-γ and the well-known IFN-γ inducible chemokine IP-10(CXCL10) are upregulated in human AA sera. Functionally relevantinflammatory cytokine/chemokine biomarkers will be identified in thesera from AA mice using a multianalyte approach (LUMINEX™ platform). Theadvantage of biomarker development in the mouse, besides genetichomogeneity, is the relative ease of longitudinal studies to identifybiomarkers that occur early or late in disease progression andconversely with treatment response. At the RNA level, C3H mouse skin IFNmicroarrays demonstrated that both Type I IFNs (IFNα₁₋₃, ε□ and type IIIFN (IFN-γ□ were upregulated as well several IFN-associated chemokines(CXCL9-11, CCL5). Cytokine/chemokines production will be examined insera using custom multianalyte LUMINEX™ assay (R&D), comprehensivelyaddressing serum levels of 9 chemokines (CCL2-5, CCL11, CXCL5, CXCL9,CXCL10, CXCL11) and 14 cytokines (IFNα, IFNγ, IL-1, IL-2, IL-4, IL-5,IL-6, IL-10, IL-12, IL-13, IL-15, IL-17, TNF, GM-CSF). We will includeantibody pairs to detect serum levels of NKG2DLs (117) and IL-15Rα.

Transcriptional Profiling of Skin:

In human AA, published studies of small series of patients have noted aType I IFN response in lesional biopsies (105) and Th1 skewing andelevated IFN-response cytokines/chemokines in the peripheral blood(106-110) and reviewed in (111). These data are mirrored in the analysisof the C3H mouse. Transcriptional profiling of RNA isolated fromalopecic mouse skin revealed a dominant IFN response. 18 of the top 21upregulated genes in AA skin were IFN response genes, which wasconfirmed by real-time PCR (FIG. 22). The upregulation of theIFN-response genes CXCL9-11 are notable since the CD8⁺NKG2D⁺ cells alsouniformly express their shared chemokine receptor, CXCR3, a receptorupregulated on short-lived immune effectors (118-120). These chemokinesare also upregulated in the sera of human AA subjects and thus can be afunctionally relevant AA skin biomarker in the skin (mRNA) and in theblood (protein). Work in this area will continue, building a database ofskin RNA signatures collected before and during ongoing treatment, thatcan be used to tailor treatment plans and as early indicators oftreatment response. Towards these goals, parallel studies will beperformed in the mouse with both intralesional steroids and CTLA4-Ig, aswell as with therapeutic interventions, to provide a transcriptionalprofile database that will be useful in cross-referencing the humantreatment studies (comparative genomic analysis between studies and forpotential clinical development of more novel approaches).

Human Subjects

Skin biopsies from the scalp will be collected from a minimum of 100Alopecia Areata patients and approximately 100 controls. The subjectpopulation will consist of AA patients and unaffected control subjectsreceiving hair transplantation. Approval for collection of these samplesunderwent expedited review/exemption since they are discarded tissue. Aprotocol for collection of scalp biopsies from AA patients is inpreparation. A minimum of 100 AA subjects will be recruited, butcollection of greater than 100 will be beneficial and increase the powerof this approach.

In order to achieve maximum homogeneity among samples for generating theexpression interactome, hair follicles will be collected exclusivelyfrom middle aged Caucasian male subjects with AA, since the matchingcontrol donors are most commonly from this group. Therefore, despite thefact that AA shows no ethnic or gender predilection, the AA patientswill be selected to match in age and gender to the controls for thepurpose of this study (middle aged Caucasian males). Patients withactive disease (ie: with some remaining hair follicles) will also beselected so that they can be efficiently microdissected.

Vertebrate Animals

Young C3H/HeJ mice (2-3 months) and retired breeders showing visiblehair loss will be acquired from Jackson Labs. The animals will be keptunder standard conditions in the animal facilities.

The mice will be administered drugs or antibodies by either systemicinjections or topically. For topical applications, the mice skin will beshaved on the dorsal surface with electric clippers. One week aftershaving, mice will receive topical application of the chemicalsdissolved in acetone or 10% v/v in propylene glycol as vehicles.Injections will be given either intraperitonealy or subcutaneously. Thedosage as well as frequency of administration will be followed as perestablished protocols. The mice will be monitored daily for signs ofdistress throughout the duration of treatment.

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Can J Gastroenterol 23, 677-683 (2009).-   95. Maticic, M., Poljak, M., Lunder, T., Rener-Sitar, K. &    Stojanovic, L. Lichen planus and other cutaneous manifestations in    chronic hepatitis C: pre- and post-interferon-based treatment    prevalence vary in a cohort of patients from low hepatitis C virus    endemic area. J Eur Acad Dermatol Venereol 22, 779-788 (2008).-   96. Kartal, E. D., Alpat, S. N., Ozgunes, I. & Usluer, G. Adverse    effects of high-dose interferon-alpha-2a treatment for chronic    hepatitis B. Adv Ther 24, 963-971 (2007).-   97. Kartal, E. D., Alpat, S. N., Ozgunes, I. & Usluer, G. Reversible    alopecia universalis secondary to PEG-interferon alpha-2b and    ribavirin combination therapy in a patient with chronic hepatitis C    virus infection. Eur J Gastroenterol Hepatol 19, 817-820 (2007).-   98. Demirturk, N., Aykin, N., Demirdal, T. & Cevik, F. Alopecia    universalis: a rare side effect seen on chronic hepatitis C    treatment with peg-IFN and ribavirin. Eur J Dermatol 16, 579-580    (2006).-   99. Yu, M. L. et al. A randomised study of peginterferon and    ribavirin for 16 versus 24 weeks in patients with genotype 2 chronic    hepatitis C. Gut 56, 553-559 (2007).-   100. Taliani, G. et al. Reversible alopecia universalis during    treatment with PEG-interferon and ribavirin for chronic hepatitis C.    J Chemother 17, 212-214 (2005).-   101. Radny, P. et al. Alopecia areata induced by adjuvant treatment    with alpha-interferon in malignant melanoma? Dermatology 209,    249-250 (2004).-   102. Agesta, N., Zabala, R. & Diaz-Perez, J. L. Alopecia areata    during interferon alpha-2b/ribavirin therapy. Dermatology 205,    300-301 (2002).-   103. Kernland, K. H. & Hunziker, T. Alopecia areata induced by    interferon alpha?Dermatology 198, 418-419 (1999).-   104. Lang, A. M., Norland, A. M., Schuneman, R. L. & Tope, W. D.    Localized interferon alfa-2b-induced alopecia. Arch Dermatol 135,    1126-1128 (1999).-   105. Ghoreishi, M., Martinka, M. & Dutz, J. P. Type 1 interferon    signature in the scalp lesions of alopecia areata. Br J Dermatol.-   106. Barahmani, N. et al. Serum T helper 1 cytokine levels are    greater in patients with alopecia areata regardless of severity or    atopy. Clin Exp Dermatol (2009).-   107. Kuwano, Y. et al. Serum chemokine profiles in patients with    alopecia areata. Br J Dermatol 157, 466-473 (2007).-   108. Arca, E., Musabak, U., Akar, A., Erbil, A. H. & Tastan, H. B.    Interferon-gamma in alopecia areata. Eur J Dermatol 14, 33-36    (2004).-   109. Benoit, S., Toksoy, A., Goebeler, M. & Gillitzer, R. Selective    expression of chemokine monokine induced by interferon-gamma in    alopecia areata. J Invest Dermatol 121, 933-935 (2003).-   110. Hoffmann, R. et al. Cytokine mRNA levels in Alopecia areata    before and after treatment with the contact allergen    diphenylcyclopropenone. J Invest Dermatol 103, 530-533 (1994).-   111. Gregoriou, S. et al. Cytokines and other mediators in alopecia    areata. Mediators Inflamm 2010, 928030.-   112. Clark, R. A. et al. The vast majority of CLA+ T cells are    resident in normal skin. J Immunol 176, 4431-4439 (2006).-   113. Clark, R. A. et al. A novel method for the isolation of skin    resident T cells from normal and diseased human skin. J Invest    Dermatol 126, 1059-1070 (2006).-   114. Bissonnette, R. et al. A randomized, double-blind,    placebo-controlled, phase I study of MEDI-545, an    anti-interferon-alfa monoclonal antibody, in subjects with chronic    psoriasis. J Am Acad Dermatol 62, 427-436.-   115. Hommes, D. W. et al. Fontolizumab, a humanised anti-interferon    gamma antibody, demonstrates safety and clinical activity in    patients with moderate to severe Crohn's disease. Gut 55, 1131-1137    (2006).-   116. Verstovsek, S. et al. Safety and efficacy of INCBO 18424, a    JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med 363,    1117-1127 (2010).-   117. Nuckel, H. et al. The prognostic significance of soluble NKG2D    ligands in B-cell chronic lymphocytic leukemia. Leukemia 24,    1152-1159 (2010).-   118. Hu, J. K., Kagari, T., Clingan, J. M. & Matloubian, M.    Expression of chemokine receptor CXCR3 on T cells affects the    balance between effector and memory CD8 T-cell generation. Proc Natl    Acad Sci USA 108, E118-127.-   119. Kohlmeier, J. E. et al. Inflammatory chemokine receptors    regulate CD8(+) T cell contraction and memory generation following    infection. J Exp Med 208, 1621-1634.-   120. Kurachi, M. et al. Chemokine receptor CXCR3 facilitates CD8(+)    T cell differentiation into short-lived effector cells leading to    memory degeneration. J Exp Med 208, 1605-1620.-   125. Ramsborg, C. G. & Papoutsakis, E. T. Global transcriptional    analysis delineates the differential inflammatory response    interleukin-15 elicits from cultured human T cells. Exp Hematol 35,    454-464 (2007).-   126. Chiossone, L. et al. Molecular analysis of the    methylprednisolone-mediated inhibition of NK-cell function: evidence    for different susceptibility of IL-2-versus IL-15-activated NK    cells. Blood 109, 3767-3775 (2007).-   127. Creed, T. J. et al. The effects of cytokines on suppression of    lymphocyte proliferation by dexamethasone. J Immunol 183, 164-171    (2009).-   128. Goleva, E., Kisich, K. O. & Leung, D. Y. A role for STAT5 in    the pathogenesis of IL-2-induced glucocorticoid resistance. J    Immunol 169, 5934-5940 (2002).-   129. Rentzos, M. et al. Circulating interleukin-15 and RANTES    chemokine in MS patients: effect of treatment with    methylprednisolone in patients with relapse. Neurol Res 32, 684-689    (2009).-   130. Tliba, O. et al. Cytokines induce an early steroid resistance    in airway smooth muscle cells: novel role of interferon regulatory    factor-1. Am J Respir Cell Mol Biol 38, 463-472 (2008).-   131. Xu, Q., Goleva, E., Ou, L. S., Li, L. B. & Leung, D. Y. CD56+    cells induce steroid resistance in B cells exposed to IL-15. J    Immunol 172, 7110-7115 (2004).

Example 5

Alopecic effector T cells were identified in the lymph node and blood ofalopecic mice using spectratyping of flow sorted cells. A blood-basedassay was developed to monitor these alopecic T cells during treatment.Cytotoxic assays have been developed for evaluating functionalcomponents of the hair follicle (HF) interactions with CTLs, in bothhuman and mice.

Using human biospecimens obtained from AA subjects, it was demonstratedthat circulating T cells express high levels of NKG2D and primary Tcells obtained from the skin are dominated by a CD8⁺NKG2D⁺ IFN-γproducing cells establishing parallels between the mouse and humandisease.

Immunophenotyping and Skin Immunobiology

To study pathways in biospecimens requires analysis of heterogeneouscell populations in the skin and in the blood. In the clinicalinvestigation of human autoimmunity, alopecia areata provides a uniqueopportunity because of the accessibility of the end-organ enablingstudy/isolation of the pathogenic immune effectors in their relevantmicroenvironment. Analytic tools and personnel required will be providedfor optimal study of these precious end-organ skin biospecimens, andtheir cellular/serological counterparts in the blood obtained from thesame individual.

Preparation and Analysis of Biospecimens:

The following will be provided to assist in the analysis of blood:

i) Multianalyte cytokine/chemokine analysis from serum samples.

ii) Flow cytometric immunophenotyping, including cytokine analysis ofhuman PBMCs from AA subjects.

iii) Flow sorting for downstream applications using T cell subsets (RNAprofiling, spectratyping).

The following will be provided to assist in the analysis of skin:

-   -   i) Preparation/analysis of viable cellular fractions of both        primary hair follicle and T cell components for functional        (cytotoxicity) and analytic studies (T cell cloning,        spectratyping).    -   ii) Immunostaining and transcriptional profiling of whole skin    -   iii) Flow cytometric immunophenotyping of dermal lymphocytes        from human and mouse AA skin.

Scientific Experiments

Pathogenic HF-specific cellular subsets in the peripheral blood of AAsubjects will be identified. To this end, spectratype analysis will beused as a tool to identify circulating human alopecic T cellspopulations, as was done for the mouse model, by matching thespectratype found in total skin T cells with the spectratypes ofspecific sorted peripheral blood T cell subsets in the peripheral bloodobtained from the same patients.

Th1-predominance in human AA will be established and the rationale andbiomarker platform for Th1-targeted therapies will be developed.Therapeutic targeting of disease-specific Th-pathways has succeeded inother human skin inflammatory diseases, most notably in psoriasis aprototypical Th17 disease. There is substantial data in the AA animalmodel for AA as a predominant Th1 disease. The Th-profile ofinfiltrating dermal AA T cells and relevant circulating T cell subsetsin the blood will be addressed using a multi-prong approach, includingmultianalyte, RNA profiling, immunstaining and intracellular flowcytometric analysis from the skin and blood.

An integrative approach to translational research begins with basicstudies identifying targetable pathways in AA, then testing thesepathways therapeutically in preclinical models, longitudinal biomarkerassessment in clinical trials and pursuing population-based researchbased on genetic studies. The pre-clinical therapeutic effects ofinterventions will be evaluated in the grafted AA mouse models,treatment effects on the inflammatory responses will be assessed in theskin and blood and human biospecimens will be provided for validation ofhuman relevance in ex vivo studies.

Monitoring the pathogenic interactions between immune cells and targetcells in the skin in humans requires sophisticated processing andanalysis of primary human tissues and blood.

Flow cytometry has become the primary tool for the identification ofcell populations according to specific parameters, and is thereforeemployed by an ever-growing number of biomedical scientists.

The immunobiology of the skin presents specific characteristics andchallenges; the special architecture of the tissue including its barrierfunctions, its associated unique immune cell populations and its closeproximity/interactions with microbial flora, and the practicaldifficulties of isolating infiltrating lymphocytes, are all part of thecomplexities needed to be understood in the approach to immunologicaldiseases of the skin. Services that provide assistance with tissueprocessing and staining, as well as cell isolation, and essential toinsuring high quality consistent results are available.

Flow cytometry is a tool for clinical investigators and a workingknowledge of its use is required for all translational immunologists.Four flow cytometers, including two workhorse instruments (FACs CANTO™and FACs CALIBER™) a 6-laser LSR II™ and a 4-laser BD INFLUX™ areavailable. The LSR II™ and BD INFLUX™ have been designed to havecomparable lasers/detectors to facilitate transition from analysis topreparative sorting. The LSR II™ is equipped with 6 lasers, capable ofdetecting in total 19 colors simultaneously providing versatility todetect both fluorescent proteins (mBanana, GFP, BFP, RFP/dsRed, mCherrymRasberry) and fluorescent dyes/chemical fluorochromes. LSR II™ cananalyze diverse cell types within heterogeneous cell populations fromtissues using a wide array of organic and inorganic fluorochromes. Theseadditional fluorescent parameters allow detection of coordinatedfunctional events in specific cell types in mixed populations (e.g.intracellular IFN-gamma, IL-10 production and phosphoprotein detectionin T cell populations from cutaneous tissues and draining lymph nodes).The basic LSR II™ instrument is equipped with three lasers; blue (488nm), red (633 nm), violet (405 nm). The custom designed LSR II™ containsin addition, three additional lasers permitting up to 20-color detectionand a 96-well plate reader. “Leave one out” 10-color panel designs for Tcell subsets (for example, Tregs), B cells and monocyte have beendesigned to take advantage of the 100 W yellow-green 594 laser which hasexquisite sensitivity for “red” ALEXA™ 594. Thus, investigators mayadopt a “leave-one out” design to sensitively examine expression for aspecific marker of interest by staining with ALEXA™ 594 conjugatedantibodies.

Quality:

Panel development for immunophenotyping lymphocyte populations in humanperipheral blood and other experiments will be conducted. Because of thecapacity of the 6 laser system of the LSR II™, a common “easy-to use”set of colors that can be used without the need for compensation wasestablished. The ability to do a 5/6-color experiment without the needfor fluorescent compensation gives increased data resolution as thefluorochromes as far apart from each other in the spectrum, and excitedindependently by each laser line. This allows for the ability ofpopulations to clearly separate, something that is rare whencompensation is applied. The current selected fluorochrome combinationis DAPI (gated on the DAPI negative cells) Pacific Blue, FITC, PE, ALEXAFLUOR™ 594 and APC, however other combinations of fluors are currentlybeing tested and used. This 5-flourochrome set serves as the baseplatform for panels, to which additional fluorochromes can be added. Ina proof-of-concept experiment, the use of 5 flurochromes was shownsimultaneously without the need for compensation. C57BL/6 splenocyteswere stained with CD8 Pacific Blue, CD3 FITC, CD45R (B220) PE, CD11bBio+Streptavidin ALEXA FLUOR™ 594, and CD4 APC.

Maintenance and Quality Control for Skin Immunobiology:

The procedure used for the isolation of lymphocytes from skin biopsieshas been adapted from a method developed by Clark and Kupper (2) inwhich biopsies are culture on 3-dimensional tantalum-coated cellfoammatrices to promote migration of T cells from the biopsies. Of eachisolated cell populations a minor fraction will be used for CD45/CD3staining to assess the number of leucocytes/T lymphocytes by flowcytometry prior to further studies or cryopreservation. For re-use, thematrices will be soaked for 30 min in 10% bleach, then rinsed in waterand transferred into a solution of ENZYTE™ enzyme cleaner (Decon Labs).The matrices will be left in this solution on a hot plate with stirrerfor 24 hrs, rinsed with distilled water and let dry before autoclaving.

Whole skin analytic techniques (e.g., immunostaining) will be provided.The following will be provided: 1) critical expertise in skinimmunobiology for the preparation/analysis of viable cellular fractionsof both primary hair follicles and cutaneous T cells; 2) biomarkerdevelopment capabilities for clinical biospecimens includingmultianalyte analysis (LUMINEX™) and transcriptional profiling ofprecious clinical biospecimens (skin and blood from subjects enrolled inclinical trials). The tools to investigate biological processes andmonitor the inflammatory AA mechanisms in pre-clinical and clinicalsamples from both the blood and the AA target end organ, the skin willbe provided.

Capabilities in microarray data analysis and storage, sequence andpathway analysis are available for the studies herein, and extend allthe way to the most recent algorithms for regulatory network reverseengineering. A number of widely-used databases are created andmaintained. In addition, all important sequence and structure databasesare maintained centrally. This allows direct large-scale searches, ifneed be using custom algorithms and cluster computing. Many of theresearch methods developed are packaged into software applications andcomputational services, freely available to the scientific community(c2b2.columbia.edu/page.php?pageid=10).

The following services are available:

1. Immunomonitoring:

-   -   a. Multianalyte Cytokine & Chemokine Analysis from Serum Samples

Two approaches are available: a) The multiplex bead based immunoassayssystems (e.g. CBA/FLOWCYTOMIX™) provides multiple analytes that allow 10or more cytokines/chemokines to be assessed on a single read of a smallvolume (50 μl) sample. The LSR II™ 96 well high throughput capacityprovides ease of use combined with efficiency. Software (“snap-to”gating) is available in house for data analysis. A multiplex bead-basedassay system will also be developed to quantify soluble human and murineNKG2DL in the sera, for which specific antibody sets are commerciallyavailable. b) Multiplex analysis using the LUMINEX™ platform isavailable in the CTSA for analysis of commercially available mouse andhuman cytokine and chemokine arrays (FIG. 24). The LUMINEX™ assays areprovided on a fee-for-service basis, with reagents purchased by theuser. Once an AA serum biomarker platform bulk purchase of selectedanalytes will enable cost-savings.

Flow Cytometric Immunophenotyping

It has been established using multiparameter flow that CD8+NKG2D+ cellsdominate the dermal leukocyte population harvested from the skin of micewith spontaneous alopecia (FIG. 25). To address the human situation,flow panels have been developed to define quantitative alterations inany of 30 PBMC subsets focusing on NK and CD4 and CD8 T cell populationsusing 3 FACS tubes (T cell subset tube, NK− marker tube for CD4, CD8 andNK cells and a third non-T cell tube); Lineage Markers: CD3, CD4, CD8α,CD8β, CD14, CD19, CD20, CD56, CD64; T cell subset Markers: CD45Ra,CD45Ro, CD56, CD62L, CCR7, CD127, FoxP3, Helio; Cutaneous ChemokineReceptor Analysis: CCR4, CCR8, CCR10, CLA, E and P-selectin; NKimmunoreceptor family members: Inhibitory: KIR/CD158, CD94/NKG2A,LILR/ILT, LAIR1 NKR-P1 Activatory: NKp46, NKp30, NKp44, NKG2C, NKG2D. Inthe initial evaluation of PBMCs from four AA subjects, increased NKG2Dexpression was observed on CD56+CD8+ T cells and NK cells, but not onCD4+ T cells (FIG. 26).

Cytokine Analysis:

Activation of freshly isolated buffy coat peripheral T cells withPMA/ionomycin will drive cytokine production by circulating memory Tcells. After a 4 hour incubation with brefeldin, cells are cell surfacemarker stained, fixed and permeabilized prior to intracellular stainingfor IL-2, IFN-γ, TNF, IL-4, IL-17, and FOXP3/helios. Thesecytokines/transcription factors together with the surface markers CD3,CD4, CD8, CD25, CD56, CD62L, NKG2D, CD45Ra, CD45Ro, CLA/CCR4 willdelineate the fraction of Th1/Th2/Th17/Tregs in the total and cutaneousCD4/CD8 naïve and memory T cell compartment and their expression ofNKG2D. Without being bound by theory, using a LSRII, which enablesmulti-parametric testing from a single tube, 2-4 million PBMCs will bemore than sufficient for this analysis. This will leave enough PBMCs(>20 million cells) from a 30 ml blood draw to provide for other goals.

Flow Sorting for Downstream Applications Using T Cell Subsets

The goal is to provide potential alopecic T cells for downstreamfunctional analysis. For example, flow sorted T cell subsets will beprovided for RNA profiling, for instance, transcriptional profiling ofNK-type T cells. For biomarker studies, honing in on the appropriatecellular subset will improve the resolution of the analysis, which willotherwise be diluted by the presence of RNA from the mixed heterogenousPBMC population.

Studies in the AA mouse indicate that the alopecic T cells (FIG. 24),are recirculating and can be readily found and isolated from the bloodand cutaneous lymph nodes. More than 5 million CD8+NKG2D+ T cells can beisolated by flow sorting from the cutaneous lymph nodes of a singlemouse. This is an impressive number of fresh primary “pathogenic” Tcells for downstream study and is a substantial improvement on the cellyields one can expect to recover from “crawl-outs” from alopecic skin.The identification of pathogenic T cell subsets in the blood of AAsubjects human AA subjects is a scientific goal. Flow isolated T cellsubsets will be provided for downstream applications including biomarkerstudies, functional studies and spectratyping to provide evidence forimmuopathogenic relevance of specific T cells subsets.

i) Spectratype Analysis of TCR Repertoire

Spectratyping or TCR β-chain length distribution analysis of RNA fromtissues or phenotypically separated lymphocyte subsets provides aqualitative portrait of the TCR clonotype utilization/repertoire of theinfiltrating T cells. In recent years this technique has been used byothers (5-7) as a highly sensitive and accurate method to delineate theproportion of clonally expanded T cells in a sample. Oligoclonality,evidence of expansion of subsets of T cells with restricted TCRs isindicative of antigenic drive in inflammatory processes and can be usedas a first step to identify pathogenic T cell clones. Since each TCRrearrangement varies in CDR3 length in multiples of three nucleotides,heterogeneity of CDR3 length within a population of T cells can be usedas a measure of TCR diversity. The principle of this technique is to PCRamplify the cDNA obtained from a T cell population using an upstreamspecific V3 family member primers and a downstream constant regionprimer that together span the CDR3 region made by combinatorial andjunctional VDJ joining. The PCR products are then fluorochrome-labelledin a primer extension (“run-off”) reaction using a fluorescently labeledconstant region primer. The products are then run on the ABI PRISM 3700DNA analyzer. With Gene Mapper software the size of the peakscorresponding to discrete CDR3 lengths can be displayed and analyzed(FIGS. 20 and 27). The data are exported and compared to referencepolyclonal repertoires, and measures of oligoclonality such as theHamming distance are calculated. The area of the peaks in each histogramreflects the expression frequency of each CDR3 length within eachfamily. A predominance of clonal expansions in the skin indicates aprimary role for dermal antigens in driving disease and will enablefocused investigation of pathogenic T cells. In contrast a polyclonalrepertoire of unexpanded clones will indicate recruitment bynon-clonally specific chemokine receptors. Comparisons of the TCRrepertoire in non-lesional skin and blood of the same individuals willindicate whether clonally expanded skin resident T cell populationsmight also be found in the circulation. Similarly sorted T cell subsetscan be used for source RNA material for repertoire analysis. Forinstance, FIGS. 20 and 27 demonstrates that in C3H mice an oligoclonalpopulation is found in the lesional skin, a repertoire that is alsoover-represented in NKG2D positive but not negative CD8 lymph nodepopulations. These data confirm that CD8+NKG2D+ T cells contain most(but not all) of the oligoclonal T cell populations found in the skin.Note that some TCR/clones are found in skin but not in drainingcutaneous lymph nodes, e.g., V3 10. These likely include CD4+ T cellpopulations found in AA skin but not present in the sorted CD8+LNfraction. This approach provides powerful evidence for “antigen-drive”,and combined with flow sorting as shown here, identifies pathogenic Tcell subsets amongst populations of cells.

This evidence of clonal sharing or oligoclonality will be investigatedand validated in mouse and human samples at the level of clonotyperesolution by preparing bacterial libraries of the PCR products usingtopoisomerase-based cloning, and sequencing the TCR 3-chain of resultingclones in a high throughput manner using 96-well based techniques asdescribed (5-7). Usually 48 or 96 clones are selected and sequenced foreach PCR product. The sequences are exported and aligned in GENEIOUS™and the fine structure of VDJ element usage and joining is determinedusing Vquest. This allows definitive identification of shared clones andlymphocyte trafficking in different regions, as well as preciseenumeration of the clonotypic composition of a sample. Furthermore,clonotypic TCRs can be used for gene transduction studies to generate Tcells and retrogenic mice expressing alopecic TCRs. This effort can alsolead to development of “humanized” transgenic TCR models of alopeciausing transgenically expressed human alopecic TCRs in HLA02 transgenicmice.

ii) Transcriptional Biomarkers of Circulating Immune Cells

A transcriptional signature marking aggressive, drug refractory Lupuscan be identified within the CD8 T cell compartment, but not when usingRNA of total PBMCs (8) Thus combining flow sorting with transcriptionalprofiling greatly powers the analytic capacity. In alopecia areata thepathogenic cellular subset in the appears to be CD8+NKG2Dhi T cells thatenvelop the hair follicle and may be of pathogenic relevance broadly inautoimmunity (9) including both celiac disease (4, 10), Type I diabetes(11) and rheumatoid arthritis (12-14). Transcriptional profiling ofthese cells in celiac disease (4, 10) previously described an NK-liketranscriptional programming of these cells that will be similarlyassessed in sort-purified circulating and skin-derived CD8 T cells. Itwill be important to specifically address the transcriptional profilesof these cellular populations to identify biological pathways central tothese cells.

Epigenetically regulated autoimmune genes using ILLUMINA™-based genomicapproaches were identified and DNA was obtained from sorted autoimmunehuman T cells from patients with Type I diabetes, alopecia areata andceliac disease. Hypermethylation of the promoter regions thatdownregulate the expression of important immunoregulatory genesincluding CD3, PD-1 and FasL was identified.

iii) Cellular Immunology

Flow cytometryic analysis of activated PBMCs can be used forintracellular cytokine analysis and or signaling activation ofphosphorylated proteins. Classic antigen specific, mitogenic or mixedlymphocyte responses can be appreciated with CFSE-stained T cells orwith thymidine incorporation. Available equipment includes a cellharvester and Microbeta/Trilux plate reader (for thymidine basedproliferation and chromium release cytotoxicity assays), ELISA platereaders/washers, ELISPOT readers.

Skin Immunobiology

Preparation of primary hair follicle and T cell components forfunctional (cytotoxicity), preparative (T cell cloning) and analyticstudies (spectratyping, RNA profiling). Biopsy specimens from AApatients on study are precious, with immunostaining and transcriptionalprofiling studies taking the highest priority given the feasibility andinformative power of these two approaches. Routinely human scalp skin isprocured from control individuals (hair transplant donors) to establishprimary cultures of individual cellular populations within the skin andhair follicle.

a) Culture of T Cells and Hair Follicle Components, Hair Follicle OrganCulture and Skin Organ Culture.

i) Hf Targets:

Human scalp skin procured from control or AA affected individuals willbe used to establish primary cultures of individual cellular populationswithin the skin and hair follicle (FIG. 28) to study the regulation ofNKG2DL in primary HF populations and to evaluate HF populations astargets in CTL assays. Interfollicular skin will be dispase treated toseparate the epidermal and dermal components and enzymatically processedto establish primary cultures of keratinocytes and fibroblasts. The hairfollicles will be microdissected to separate the mesenchymal componentsand further used to culture dermal sheath cells (DSC) and dermal papillacells (DPC). Serum free hair follicle organ culture established in thelaboratory modeled after protocols from Kondo and Philpott et al (15)(FIG. 30) will be used to routinely culture individual follicles fromcontrol individuals, as well as lesional (when possible) and controlskin from AA patients. The follicles are cultured in serum free growthconditions and show normal anagen growth for 7-10 days, followed bycatagen entry and disintegration of the matrix (16). For cytotoxicityassays, the follicles will be transferred to media which sustainscytotoxic T-Cells or NK cells (MYELOCULT™, Stemcell Technologies) for4-8 hrs. The skin explants maintain normal skin architecture, as well asthe immune microenvironment, and will be used for functional studies(17).

ii) T Cell Effectors:

Isolation of sufficient numbers of T cells from enzymaticallydigested/dispersed human skin can be difficult; however cultured T cellswerent expanded using the dermal crawl-out approach developed by Clark(2). Skin explants have been used to expand the resident T-cellpopulation of the skin by culturing the explants on cell foam matricesfor 3 weeks in IMDM containing 20% FCS, IL-2 and IL-15. This techniqueyields 0.3-3.0×106 T cells per 4 mm biopsy enabling immunophenotyping, Tcell cloning, Th-profiling, and other downstream applications (e.g.,transcriptional profiling, cytotoxicity and other functional assays).For AA patients on intervention trials, if sufficient T cells areobtained after three weeks of expansion (>1×10⁶) they will be divided,with half the population used for RNA isolation/profiling and the otherhalf used for flow immunophenotyping/Th profiling. If the yields areless than 1×106 total cells, studies will be limited to RNA profiling.For AA patients that are not on study, there will be more latitude forusing crawl-out T cells and autologous HF targets for alternativefunctional studies, including for instance cytolyic assays and studieswith antibody blockade/small molecules. As an example of the ability toanalyse rare clinical populations of T cells, FIG. 29 illustrates a Tcell “crawl-out” assay, in which cultured human dermis obtained frompunch skin biopsies are used as source material for flow cytometricanalysis.

Note that T cells obtained from AA skin biopsies are oligoclonal IFN-γ□producing CD8+NKG2D+ T cells in striking contrast to the expectedpolyclonal CD4 population seen in crawl-outs from normal skin (2, 18).

b.) Interaction of the Skin and the Immune Components in Alopecia AreataModels

Established protocols are available for assessing skin-immuneinteractions for the studies herein. Primary cultured cells, as well asorgan-cultured follicles, can be used as targets using cytotoxic T cellsfrom control or patient peripheral blood lymphocytes, or those derivedfrom the skin (FIG. 29). The immune effector cells can be co-culturedwith CFSE-labeled target cells/hair follicles and the cytolysis will bemeasured colorimetrically by LDH release, or alternatively, the deadcells will be stained with 7-AAD and the cell numbers counted by flowcytometry. These assays can be used to determine molecular requirementsfor the interaction of immune effectors and HF targets, includingcytotoxicity. For instance as shown here, cytotoxicity required NKG2Dengagement and prior sensitization of targets with cytokines/TLR ligandsknown to upregulate NKG2DL transcription and surface expression.

Evaluation of Skin Biomarkers of Pathogenesis: Immunostaining andTranscriptional Profiling

AA shows high correlation with several autoimmune disorders, making thehair follicle a highly accessible organ in which to study basicmechanisms of autoimmunity. There has been substantial interest inidentifying early surrogate biological markers of pathogenesis. As such,AA represents a model system for biomarker development which may haverelevance to a broad range of diseases. The following biomarkers arecurrently used to monitor AA development and response to treatment bothin humans and in mice.

a) Immunohistochemical Staining for Lymphocytic Infiltration.

AA is associated with the presence of intrafollicular, andparafollicular immune infiltrate. For immunohistochemistry, tissue willbe either fixed in 10% formalin in PBS for 8 hours at room temperatureand stored in 70% ethanol for paraffin sections or embedded directly inCRYOMATRIX™ (Shandon, Waltham, Mass.) on dry ice for frozen sectionswhich are stored at −80° C.

Paraffin-embedded tissue blocks are cut into 8-m sections, andhematoxylin and eosin (H&E) staining is carried out for histologicalstudies. Frozen tissue is also sectioned to a thickness of 8 □μm andfixed in 4% paraformaldehyde for immunofluorescence staining. Sectionsare stained with fluorescence-labeled secondary antibodies andimmunofluorescence imaging carried out using Zeiss Axioskop microscope.Basic immunohistological/fluorescent staining will include staining withprimary antibodies to CD3, CD4, CD8. As an example of the immunostains,evidence that MHC I and II and ICAM-1 are massively upregulated inalopecic HFs which are associated with a dense CD8 dominated infiltrateis provided (FIG. 31).

b) Isolation of Total Skin RNA and Quantitative PCR for InflammatoryMarkers.

The human and mouse skin will be tested for elevation of inflammatorygene transcripts already defined, including IFN-response genes, and genesignatures to be defined, including “NK-type” CD8 T cell transcripts.Total RNA will be extracted from skin using an RNeasy purification kit(Qiagen). DNase-treated total RNA will be reverse-transcribed usingSuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif.).

RT-PCR shall be performed using SYBR™ Green Master Mix and an ABI PRISM7000™ Sequence Detection System (Applied Biosystems, Foster City,Calif.). GAPDH and -actin will be used as internal normalization controlgenes.

One of the goals of the studies herein is to establish the phenotype ofcirculating AA effector populations by matching spectratypes of sortedperipheral blood AA T cell subsets with those found in total skin Tcells from the same patient. Flow cytometric sorting techniques will becombined with spectratype analysis to identify pathogenic T cells in thecirculation of AA patients. This same approach was used in the mousemodel to show that CD8+NKG2D+ lymph node cells contain the majority ofthe T cell clones found in alopecic skin. In AA subjects, the TCRrepertoire will be compared in lesional and non-lesional skin and bloodof the same individuals to identify whether clonally expanded skinresident T cell populations are also be found in the circulation. Byprogressive refinement of the sort criteria to isolate source RNAmaterial from circulating T cell subsets one should be able to (at leastpartially) match the TCR repertoire found in total skin from the samepatient and in this way identify the immunophenotypic markers ofpathogenic CD4 and CD8 T cells. Identification of pathogenic T cellsubsets will be enormously valuable for monitoring clinical studies,refining biomarker development and would provide unique cellularmaterials for bedside-to-bench studies of pathogenesis.

Another goal of the studies herein is to establish the Th profile of AAT cells infiltrating the dermis and circulating in the peripheral blood.Pathogenic AA T cell Effector Differentiation/Cytokine Profile:Therapeutic targeting of specific Th-pathways has succeeded in otherhuman skin inflammatory diseases, most notably in psoriasis aprototypical Th17 disease. There is substantial data in the AA animalmodel for AA as a predominant Th1 disease (19-23). To establishTh1-predominance in humans and develop the rationale and biomarkerplatform for Th1-targeted therapies, the Th-profile of infiltratingdermal AA T cells and relevant circulating T cell subsets in the bloodwill be addressed using a multi-prong approach including multianalyte,RNA profiling, immunostaining and intracellular flow cytometric analysisfrom the skin and blood. Refining the immunophenotypic markers for AACD4 and CD8 T cells will of course allow detection of Th-profiles of thepotential alopecic-specific T cells amongst the polyclonal circulatingpopulation.

Although transcriptional profiling of AA lesions from a small series ofsubjects are indicative of Th1-type skewing (24), without being bound bytheory, AA is not “one disease”. The common occurrence in AA subjects ofco-morbid immune conditions that reflect both an underlying Th2 orTh1/17 bias that predisposes to diseases with common pathogenicpathways. including hypersensitivity (25) (dermatitis) and otherautoimmune conditions (26-33) (see studies described herein) suggeststhat there may be a Th2-type AA as well as a Th1 or Th17 type of AA.Thus it is important to identify functional T cell responses in PBMCsfrom the periphery and from AA lesional tissue explants from a largenumber of patients that might identify subsets of AA with different Thprofiles and to correlate these immune function biomarker parameterswith the presence or absence of the relevant SNPs.

The T cell immunophenotype of peripheral T cells and lesional T cellswill be assessed. 50 AA patients and 50 normal controls will be studiedfrom the GWAS, with attached clinical history. Assessment of Th-profilesof total circulating T cells is straight forward but multi-parametricflow will be applied to T cell subsets for more granular assessment, asaided ultimately by definition of immunophenotypic markers of AApathogenic populations. Isolation of sufficient numbers of T cells fromenzymatically digested/dispersed human skin can be difficult, and maybealtered by culture conditions. Correlation will be performed withtranscriptional profiling using whole skin approaches and microdissectedskin samples to describe the cytokine profile within the follicularsheath infiltrate as well as from infiltrates in the interfollicularepithelium.

Human Subjects

Skin biopsies from the scalp will be collected from normal subjectsconsist of AA patients and unaffected control subjects receiving hairtransplantation. Additional biopsies will be obtained from subjects onclinical trials.

The sources of research material will be scalp skin biopsies from AApatients and controls. RNA from small tissue samples, includingmicrodissected hair follicle compartments are routinely obtained. Tissuebiopsies from AA patients and controls will be provided.

Vertebrate Animals

Young C3H/HeJ mice (2-3 months) and retired breeders showing visiblehair loss will be acquired from Jackson Labs. The mice strainsestablished by each participating principle investigator will be housedin their respective animal housing suites. Mice will be given freeaccess to water and pellet diet (5010 rodent diet, LabDiet, PMINutrition International).

Primary cell culture for individual skin and follicle components as wellas hair/skin organ cultures will be established for wild-type andtransgenic or knock-out mice. These studies also involve hair follicleand skin tissue characterization. The mice will be euthanised by CO2asphyxiation followed by cervical dislocation, a veterinary-approved andwidely recommended method. It is the quickest and most humane methodthat is not associated with any pain or stress for the animals. Micewill be shaved on the dorsal surface with electric clipper and then skintissue will be dissected out. The isolation scheme of individualcellular components of the skin and hair follicle as well the T-cellsisolation from skin explants is described in FIGS. 25, 28 and 29. SkinSamples will also be embedded in OCT and paraffin. In addition to skin,spleen and thymus will also be dissected from the animals and furtherused for flow cytometry. Serum from the animals will also be collectedby tail bleeding and used for cytokine profiling.

REFERENCES

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Hirota, R., Tajima, S., Yoneda, Y., Okada, M., Tashiro, J.,    Ueda, K., Kubota, T., and Yoshida, R. 2003. Induction of hair    regrowth in the alopecia site of IFN-gamma knockout mice by    allografting and IFN-gamma injection into the transplantation site.    J Interferon Cytokine Res 23:433-439.-   21. Gilhar, A., Kam, Y., Assy, B., and Kalish, R. S. 2005. Alopecia    areata induced in C3H/HeJ mice by interferon-gamma: evidence for    loss of immune privilege. J Invest Dermatol 124:288-289.-   22. Skurkovich, S., Korotky, N. G., Sharova, N. M., and    Skurkovich, B. 2005. Treatment of alopecia areata with    anti-interferon-gamma antibodies. J Investig Dermatol Symp Proc    10:283-284.-   23. Nakamura, M., Jo, J., Tabata, Y., and Ishikawa, O. 2008.    Controlled delivery of T-box21 small interfering RNA ameliorates    autoimmune alopecia (Alopecia Areata) in a C3H/HeJ mouse model. Am J    Pathol 172:650-658.-   24. Subramanya, R. D., Coda, A. B., and Sinha, A. A. 2010.    Transcriptional profiling in alopecia areata defines immune and cell    cycle control related genes within disease-specific signatures.    Genomics 96:146-153.-   25. Barahmani, N., Lopez, A., Babu, D., Hernandez, M., Donely, S.    E., and Duvic, M. 2009. Serum T helper 1 cytokine levels are greater    in patients with alopecia areata regardless of severity or atopy.    Clin Exp Dermatol.-   26. Chu, S. Y., Chen, Y. J., Tseng, W. C., Lin, M. W., Chen, T. J.,    Hwang, C. Y., Chen, C. C., Lee, D. D., Chang, Y. T., Wang, W. J., et    al. 2011. Comorbidity profiles among patients with alopecia areata:    The importance of onset age, a nationwide population-based study. J    Am Acad Dermatol.-   27. Du Vivier, A., and Munro, D. D. 1975. Alopecia areata,    autoimmunity, and Down's syndrome. Br Med J 1:191-192.-   28. Goh, C., Finkel, M., Christos, P. J., and Sinha, A. A. 2006.    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Increased risk for type I    (insulin-dependent) diabetes in relatives of patients with alopecia    areata (AA). Am J Med Genet 51:234-239.-   33. Yano, S., Ihn, H., Nakamura, K., Okochi, H., and    Tamaki, K. 1999. Antinuclear and antithyroid antibodies in 68    Japanese patients with alopecia areata. Dermatology 199:191.

Example 6

In vivo pre-clinical studies of blocking Jak1/Jak2 was performed usingthe INCB018424 Jak1/Jak2 inhibitor in the grafted animal model of thedisease, C3H-HeJ mice. 0/5 mice developed alopecia after treatment withINCB018424, whereas 2/5 placebo treated mice developed AA.

Example 7—Mouse Transplant Model for Alopecia Areata

Alopecia areata (AA) is one of the most prevalent autoimmune diseasesand manifests as nonscarring hair loss. The course of the disease isunpredictable, and there are currently no consistently efficacioustreatments available. Although research into disease pathogenesis andthe development of targeted therapies are lacking, the creation of amouse transplant model with a high incidence of disease has opened upnew avenues of preclinical experimentation.

Several lines of evidence support the potential efficacy of JAK3inhibitors in the treatment of AA: (1), a recently completed genome-wideassociation study of AA identified a number of gene-associated loci withimmunological relevance, including those for IL-2/IL-2RA and IL-21; (2)initial findings in a preclinical mouse model suggested disruption ofIL-15/IL-15R signaling ameliorates the disease; and (3) data indicatethat T cells, dependent on IL-7 and IL-15 for survival, mediate thedisease in the mouse model and in humans.

We therefore assessed the efficacy of JAK3 inhibition on the developmentof alopecia in a preclinical mouse model. Alopecic C3H/HeJ skin graftswere transplanted onto unaffected C3H/HeJ mouse recipients andsubsequently treated with a JAK3 inhibitor administered by osmotic pump,vehicle administered by osmotic pump, or PBS injections. While 4/4PBS-treated and 2/2 vehicle-pump treated mice developed alopecia by sixweeks following transplantation, 0/5 mice treated with the JAK3inhibitor developed alopecia. Microarray analysis of skin,immunohistochemical stains of skin for T cell infiltrate, and serummarker assessments demonstrated decreased inflammatory profiles in micetreated with JAK3 inhibitors. In total, our findings suggest JAK3inhibitors may be an effective treatment in patients with AA.

Example 8—Targeting the Immune Effector Response in AA

Specific autoimmune mechanisms underlying Alopecia Areata (AA) haveremained obscure and therefore clinical investigation of AA hashistorically lagged behind other autoimmune diseases.

Identify Effective, Clinically Relevant, Therapies in Mouse Models ofAlopecia Areata.

In both human AA and in the AA mouse model, local hair follicle (HF)IL-15/NKG2DL upregulation was identified as key inflammatory signalsthat recruit/activate CD8⁺ T cells, likely the critical AA immuneeffectors responsible for IFN-gamma (γ) production and hair follicle(HF) cytotoxicity. These observations provided the rationale for thestudies to therapeutically target the IL-15/NKG2D.

It has been shown that the IL-15 pathway can be blocked using JAK3protein tyrosine kinase inhibitors (PTKi).

Small Molecule PTKi Prevents AA.

5 mice were treated systemically with tofacitinib (CP-69055; 10mg/kg/day within an Alzet pump). Tofacitinib is a JAK3 inhibitor knownto inhibit IL-15 signaling in human whole blood assays. None of thetreated mice developed alopecia areata or cutaneous lymphadenopathy,whereas untreated mice manifested both AA and associated cutaneouslymphadenopathy (FIG. 32). Topical delivery approaches will be pursuedfor AA reversal in the mouse model that has the major benefit of animproved therapeutic index for clinical development.

Identify Disease-Associated AA Biomarkers and their Reversal withEffective Therapy in C3H Mice.

Cellular, inflammatory, and molecular biomarkers have been assessed intreated mice. Targeted therapy with tofacitinib eliminated thepathogenic CD8⁺NKG2D^(hi) cells from both the skin and the cutaneousdraining lymph nodes and moreover down-regulated inflammatory biomarkersin the skin, including MHC molecules and the AA-associated IFN signature(FIG. 33). HF NKG2DL expression was reduced although not completelyeliminated (e.g. H60 expression by immunostain) (FIGS. 33 and 34),indicating H60 upregulation as a proximal event.

Transcriptional profiling analysis of treated vs. untreated skin showsabrogation of the cutaneous IFN-signature with treatment. These murinetranscriptional profiles will be integrated computationally withtranscriptional profiles obtained from human skin biopsies fromuntreated alopecic subjects to identify candidate dynamic humanbiomarkers that are associated with patchy stable disease vs.progressive disease vs. alopecia universalis.

Interpretation of PTKi Targeted Approaches.

PTKi's vary greatly in their selectivity of their target kinases. Fortofacitinib, JAK sensitivity is greater for JAK3>JAK1>>JAK2 (IC₅₀ is28-50 nM, 140-180 nM, 1000-5000 nM, in whole blood assays,respectively). In the treated mice, tofacitinib serum levels would be30-40 nM, and potential impacts will be assessed on both JAK1 and 3activity. For ruxolitinib (a JAK1/2 inhibitor), sensitivity isJAK2>JAK1>>JAK3 (IC₅₀ is 3.3 nM, 2.8 nM, and 428 nM for in vitro kinaseinhibition, respectively). Pharmacodynamic JAK inhibition in the mouseis likely to be transient since ruxolitinib dosing is once daily and thehalf-life in rodents is 3-6 hours.

For example, a Jak3 inhibitor can exert selective inhibition activity ofits JAK3 target at about 10 nM, about 15 nM, about 20 nM, about 25 nM,about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, or atabout 55 nM. A Jak3 inhibitor can also exert selective inhibitionactivity of its JAK3 target at about 60 nM, about 65 nM, about 70 nM,about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about100 nM, about 105 nM, about 110 nM, about 115 nM, about 120 nM, about125 nM, about 130 nM, or at about 135 nM.

For example, a Jak3 inhibitor can also exert inhibition activity of itsJAK3 target, while also able to promiscuously exert inhibition activityof a JAK1 target, at about 140 nM, about 145 nM, about 150 nM, about 155nM, about 160 nM, about 165 nM, about 170 nM, about 175 nM, about 180nM, about 185 nM, about 190 nM, about 195 nM, about 200 nM, about 205nM, about 210 nM, about 215 nM, about 220 nM, or at about 225 nM.

For example, a Jak3 inhibitor can also exert inhibition activity of itsJAK3 target at about 250 nM, about 300 nM, about 350 nM, about 400 nM,about 450 nM, about 500 nM, about 650 nM, about 700 nM, about 750 nM,about 800 nM, about 850 nM, about 900 nM, or at about 950 nM.

For example, a Jak3 inhibitor can also exert inhibition activity of itsJAK3 target, while also able to promiscuously exert inhibition activityof a JAK2 target, at about 1000 nM, about 1250 nM, about 1500 nM, about1750 nM, about 2000 nM, about 2250 nM, about 2500 nM, about 2750 nM,about 3000 nM, about 3250 nM, about 3500 nM, about 3750 nM, about 4000nM, about 4250 nM, about 4500 nM, about 4750 nM, about 5000 nM, about5250 nM, about 5500 nM, about 5750 nM, about 6000 nM, about 6250 nM,about 6500 nM, about 6750 nM, about 7000 nM, about 7250 nM, about 7500nM, about 7750 nM, or at about 8000 nM.

For example, a Jak1/2 inhibitor can exert selective inhibition activityof its JAK1 and/or JAK2 target at about 0.5 nM, about 1.0 nM, about 1.5nM, about 2.0 nM, about 2.5 nM, about 3.0 nM, about 3.5 nM, about 4.0nM, about 4.5 nM, about 5.0 nM, about 5.5 nM, 6.0 nM, about 6.5 nM,about 7.0 nM, about 7.5 nM, about 8.0 nM, about 8.5 nM, about 9.0 nM,about 9.5 nM, or about 10.0 nM. For example, a Jak1/2 inhibitor canexert selective inhibition activity of its JAK1 and/or JAK2 target atabout 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM,about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about95 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300nM, about 350 nM, or at about 400 nM.

For example, a Jak1/2 inhibitor can also exert inhibition activity ofits JAK1 and/or JAK2 target, while also able to promiscuously exertinhibition activity of a JAK3 target, at about 425 nM, about 450 nM,about 475 nM, about 500 nM, about 525 nM, about 550 nM, about 575 nM,about 600 nM, about 625 nM, about 650 nM, about 675 nM, about 700 nM,about 725 nM, about 750 nM, about 775 nM, about 800 nM, about 825 nM,about 850 nM, about 875 nM, about 900 nM, about 925 nM, about 950 nM,about 975 nM, or at about 1000 nM.

To activate a Jak protein target in the skin, the skin should bepenetrated at about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about2.5 mm, about 3 mm, about 3.5 mm, or at about 4 mm. Depending on thedepth of penetration, the concentration of a JAK inhibitor, such as aJAK1/2 inhibitor or a JAK3 inhibitor, can range from about 25 nM toabout 5000 nM. At the higher concentration ranges, one JAK inhibitor canelicit an inhibitory effect not only of its target but also of other JAKproteins.

Integration of Human and Mouse Gene Expression Profiles.

The ribbon plots shown in FIG. 35 depict microarray data of normalizedskin expression profiles of select genes from mice treated with 13 weeksof the JAK3 inhibitor CP690550 and PBS control. Briefly, after 13 weeksof treatment with the JAK3 inhibitor, skin was harvested and subjectedto microarray analysis and compared with that of mice treated with PBS.The data show treatment-related elimination of the IFN-responsechemokines and markers of CD8 cytotoxic effectors. Expression arraysfrom mouse and human will be integrated to compare expression profilesand eventually response to treatment.

Example 9—Systemic Treatment with JAK3i

“NK-type” CD8αβ+ T cells are massively expanded in alopecic skin anddraining LNs, and are required for T cell mediated transfer implicatingthis cytotoxic cell subset as the likely pathogenic effectors.

IL-15 is a key cytokine responsible for inducing CD8 T effectors invitro. Moreover, IL-15 is produced by intestinal epithelial cells and isa known precipitant of CD8 cytotoxicity in celiac disease.

Alopecia areata is marked by IL-15/IL-15Ra upregulation in the human andmurine hair follicle and implicates this cytokine as an end-organtrigger of CD8-mediated autoreactivity in AA.

Systemic IL-15 blockade with a small molecule JAK3 PTKi effectivelyprevented alopecia areata, eliminating expansion of the pathogenicNK-type CD8 population and ablating the inflammatory signature in theskin. FIG. 53 shows reversal of established disease with topicaltherapies.

Example 10—Cellular Identity of Cytotoxic T Lymphocytes in AlopeciaAreata Defines a Therapeutic Strategy

Alopecia areata (AA) is a common autoimmune disease resulting in animmune attack on the hair follicle¹. Although T cells have beenimplicated in the disease process, the pathways underlying theiractivation had not been determined². Prior to the GWAS study, thegenetic basis of AA was largely undefined. Unexpectedly, a region ofstrong association was identified within the ULBP gene cluster onchromosome 6q25.1, encoding activating ligands of the natural killercell receptor, NKG2D, which had never before been implicated in anautoimmune disease. Guided by the GWAS studies implicating NKG2D ligands(NKG2DL)³, here ‘NK-type’ CD8+ T cells were identified as the dominanteffectors, which are both necessary and sufficient for diseaseinduction. Global transcriptional profiling of mouse and human AA skinrevealed striking signatures indicative of a robust IFNγ response andthe presence of a cytotoxic T cell infiltrate.

Using the graft model of C3H/HeJ mouse skin to transfer AA, diseaseprevention can be recapitulated when treating at the time of grafting,as well as reversal of established disease by allowing grafted mice tofirst lose their hair. Systemically-administered pharmacologicalinhibitors of the JAK3 protein tyrosine kinases eliminated theIFN-signature and prevented the development of AA, and topicaladministration reversed established disease. Notably, these effects weredurable up to 3 months after cessation of therapy. These findingsillustrate the power of GWAS studies in uncovering new diseasemechanisms, which have rapidly translated into new therapeuticopportunities in AA.

AA is a T cell mediated autoimmune disease characterized by hair lossand, histologically, by infiltrating T cells surrounding the hairfollicle bulb, the so-called “swarm of bees^(1,2). Previous studies haveshown that transfer of total T cells (but not B cells or sera), canconfer the disease in human xenograft models as well as the C3H/HeJmouse model^(4,5). However, the identity of the specific pathogenic Tcell subsets in either human or mouse AA has not yet been defined.

Guided by a previous GWAS study, which identified ULBP3/6 as the mosthighly significant non-HLA risk locus in AA (p=2×10⁻²⁰)³, a focus hasbeen centered on establishing NKG2DLs as potential “danger signals” inthe disease process. Upregulation of ULBP3 has previously shown in humanAA hair follicles, associated with a dense infiltrate of CD8⁺NKG2D⁺ Tcells³. Here, the role of these cells in AA immunopathogenesis, as wellas a target for potential therapeutic intervention, was investigated.

To determine the mechanistic importance of these cells in pathogenesis,the C3H/HeJ mouse model of AA was used, in which spontaneous diseasedevelops with ˜15% incidence between 6 and 12 months of age⁶, and thegraft-transfer C3H model was used, in which grafts from affected animalscan transfer disease to 100% of C3H/HeJ recipients in 8-12 weeks time⁷.Lesional skin biopsies revealed that CD8⁺NKG2D⁺ T cells infiltrate theepithelial layers of the hair follicle that overexpress the NKG2DLs H60and Rae-1, analogous to the situation in human AA (FIG. 54A-B and FIG.58A-B). Flow cytometric examination of the CD45⁺ leukocyte population inthe skin identified a striking expansion of CD8⁺NKG2D⁺ T cells (FIG.54D). Other cell types, including CD4⁺ T cells were present in muchsmaller numbers (FIG. 58C). C3H/HeJ mice exhibit striking cutaneouslymphadenopathy (FIG. 54C) with increased total cellularity and, asnoted in the skin, a dramatic expansion of a CD8⁺NKG2D⁺ cell populationnot present in disease-free C3H/HeJ mice (FIG. 54C-D).

The immunophenotype of the skin-infiltrating CD8⁺ T cells were similarto the CD8⁺NKG2D⁺ population found in the cutaneous lymph nodes: CD8ab⁺effector memory T cells (CD8^(hi)CD44^(hi)CD62L^(low) CD103⁺) bearingseveral NK immunoreceptors, including CD49b and NKG2A/C/E (FIG. 54E andFIG. 58D). These ‘NK-type’ CD8⁺ T cells expressed high levels of IFNgand exhibited NKG2D-dependent cytotoxicity against ex vivo expandedsyngeneic dermal sheath target cells (FIG. 54F). Gene expressionanalysis of the CD8⁺NKG2D⁺ T cells isolated from alopecic C3H/HeJ lymphnode cells using RNAseq demonstrated a transcriptional profilecharacteristic of effector CTLs as defined by the ImmGen Consortium⁸⁻¹⁰and identified several additional “NK-specific” transcripts (FIG. 54Gand FIG. 59 and Table 14).

TABLE 14 Differentially expressed genes in CD8 + NKG2D + memory T cellsvs. CD8⁺NKG2D⁻ memory T cells isolated from cutaneous lymph nodesobtained from alopecic mice. RNAseq was performed on cRNA made isolatedfrom flow-sorted cells (BD influx) from total cutaneous lymph nodes fromtwo alopecic mice. GeneSymbol FC GeneSymbol FC GeneSymbol FC Gstm5 63.996430531B16Rik 3.63 Tmem151a 2.36 Gzma 52.13 Marco 3.62 Ly6d 2.36 Ifitm144.81 Sulf2 3.61 Irf5 2.35 Cx3cr1 38.24 Reep1 3.61 Bhlhe40 2.35 Sprn35.20 Anxa4 3.61 Zfp608 2.34 Tjp1 34.76 Procr 3.60 Fam184a 2.33 Igsf532.13 Slc40a1 3.60 Mafb 2.33 Tmem132e 31.71 Tnfrsf8 3.59 Coch 2.32Cmklr1 30.91 Matk 3.56 Gpr179 2.32 5430421N21Rik 30.69 Cysltr2 3.56Ffar1 2.32 Csf1 29.97 Smtn 3.55 H2-Ob 2.32 Olfr60 28.79 Rasl11b 3.54Nav2 2.32 4933431E20Rik 27.87 Amph 3.52 Srpk3 2.31 Metrnl 27.44 Cysltr13.52 Cd209b 2.31 Epdr1 27.43 Klri1 3.52 Arhgef12 2.31 Cd244 27.22B4galt2 3.51 Arvcf 2.31 Gpr141 26.45 Mlf1 3.49 Serpina3g 2.30 Ccr1 26.17B4galt4 3.49 Ppp1r3f 2.30 Ccr8 23.75 Slc4a11 3.47 Cdc25c 2.30 Pkd2 23.23Htra1 3.47 Tmem159 2.30 Lgals3 22.66 Ebf1 3.44 Atcay 2.30 Esm1 21.59Mpp2 3.44 Sept4 2.29 AI661453 20.69 Ica1 3.44 Cacnale 2.29 Sema6d 20.58Tigit 3.43 Plxna1 2.29 Tmem171 19.13 Lzts1 3.43 Fzd6 2.29 Havcr2 19.01Epha3 3.43 Zcchc24 2.29 Rasgrf1 18.32 Mrgpre 3.43 Ttbk1 2.28 Bean1 18.25Vdr 3.42 Cd40 2.28 Adora3 18.13 Npnt 3.41 Atp6v0a1 2.28 Itga1 17.66D630039A03Rik 3.40 Gnaol 2.27 Tpbg 17.52 Rapgef3 3.37 Gsg2 2.27 Gzmb17.43 Cnksr1 3.37 Dock5 2.26 Gpr44 17.11 Napsa 3.37 Tlr9 2.26 Stk32c16.81 E030011O05Rik 3.37 Fbxo6 2.26 Bcar1 16.72 1700019E19Rik 3.372410022L05Rik 2.26 Cntn1 16.70 Ncald 3.37 Ltbp4 2.25 Slc38a8 16.65 Ccl83.36 Ncf2 2.25 Adam8 16.15 Dhrs3 3.36 Cela1 2.25 Usp44 15.85 Ppap2b 3.36Ncr1 2.25 B4galnt4 15.50 Gas2l1 3.36 Irf4 2.25 Cass4 14.94 St3gal2 3.35Bank1 2.24 Galr3 14.86 Phactr2 3.34 Ell2 2.23 Ifitm2 14.77 Rasd1 3.341190002F15Rik 2.23 Rimbp2 14.64 Mafa 3.33 Unc5b 2.23 Fam171b 14.02Olfr433 3.33 Bspry 2.23 Srxn1 13.78 L1cam 3.31 D17H6S56E-3 2.23 Ptger313.70 Pik3r6 3.31 Chst2 2.23 Lrat 13.48 C1qa 3.31 Slc41a3 2.22 Trpm613.43 Fgd1 3.30 Ctla2b 2.22 Lrrn2 13.17 Gm17745 3.30 Gas2l3 2.22 Arnt213.06 Apol9b 3.30 Casp4 2.22 Yap1 13.04 Cmtm7 3.29 Dcxr 2.21 Anxa1 12.85Bicd1 3.29 Ndrg4 2.21 Gpr56 12.78 Rgs2 3.29 Muc1 2.21 Rai14 12.59 Nos1ap3.29 Lass6 2.21 Lypd3 12.52 Bcl2a1d 3.29 Pls3 2.21 Gm5127 12.23 Eif4e33.28 Dok3 2.20 Pdgfrb 12.13 Sgtb 3.28 Carhsp1 2.20 Tktl1 11.87 Fut7 3.28Ppfibp1 2.20 Gzmk 11.63 Kif19a 3.27 Stxbp1 2.20 Olfr525 11.61 Mtmr7 3.27Ctsh 2.20 AF529169 11.57 Fam114a1 3.27 Edaradd 2.20 Olfr523 11.48 Sdc43.27 Galnt10 2.20 Selm 11.43 Farp1 3.26 Prr5 2.19 Sema4c 11.25 Fbxl213.25 Rcbtb2 2.19 Ppp2r2c 11.12 Ccrl2 3.25 Pik3c2b 2.19 Fxyd4 11.09 H1fx3.24 Nqo2 2.19 Sept5 11.07 Eif2c4 3.24 Fbxo30 2.18 Styk1 11.04 Ube2l63.23 Mki67 2.17 Myadm 10.84 Gpr68 3.23 Prnp 2.17 Wnt10b 10.654930544D05Rik 3.23 Ahr 2.16 Fgl2 10.61 Hrh4 3.22 C030034L19Rik 2.16Gcnt4 10.59 Ryr1 3.22 Lgals1 2.16 Emp1 10.44 9030418K01Rik 3.22 Gm111102.15 Cacng8 10.38 Acot11 3.22 Jup 2.15 Car5b 10.36 Pon3 3.22 Fam20a 2.15Ly6g5b 10.32 Serpine2 3.20 Sfpi1 2.15 Lmna 10.16 Ly6a 3.20 Lrfn4 2.14Vipr2 10.08 Lrrn3 3.19 Cdkn2c 2.14 Ptpn5 10.01 B3galt5 3.19 Crybg3 2.14Plekhf1 9.92 Ankrd29 3.19 Trerf1 2.14 1113 9.87 Ccl1 3.19 Dok4 2.149430020K01Rik 9.87 Serpina3f 3.19 Cdk6 2.13 Klrk1 9.80 St14 3.15 S1pr32.13 Itsn1 9.65 Dscam 3.14 Rxra 2.13 Ret 9.62 Fam129a 3.14 Cd163l1 2.12Itgae 9.58 Med12l 3.14 Pwwp2b 2.12 Col6a3 9.50 1300014I06Rik 3.131700017B05Rik 2.12 Nbea 9.41 Rnf216 3.13 2310010M20Rik 2.12 Rab27b 9.41St3gal5 3.13 Nek2 2.12 Clec12a 9.36 Slc35e4 3.12 Lix1l 2.12 Mgat3 9.35Rab3il1 3.12 Ggta1 2.11 Zfp57 9.30 Tmem176a 3.12 Rorc 2.11 S100a4 9.27Syngr3 3.12 Fosb 2.11 AI414108 9.22 Il9r 3.12 Rimkla 2.11 Trim58 9.19Lrrk2 3.11 Txlnb 2.10 Irak3 9.18 Zfp532 3.11 Smox 2.10 Gpr34 9.15 Ache3.10 Tcf4 2.10 Mt3 9.04 Wipf3 3.09 Fgd2 2.10 AW555464 9.02 Cdkn1a 3.09Cd300lf 2.10 Perp 9.00 Ccdc136 3.08 Map3k13 2.10 Ace 8.94 Epn2 3.07Neil3 2.09 Tmeff2 8.92 Fam167a 3.07 Optn 2.09 Slc35f3 8.81 Syp 3.07A930038C07Rik 2.09 Dyrk3 8.81 Spry2 3.06 Klra5 2.08 Nxnl2 8.72 Tiam13.06 Cyp4f18 2.08 Galnt3 8.67 Gnaq 3.05 Akapl7b 2.08 Cish 8.63 Armcx63.05 2700081O15Rik 2.07 Vash1 8.58 Raph1 3.05 Pcyoxl1 2.07 Klre1 8.58F730043M19Rik 3.05 Cd226 2.07 Dmrt2 8.56 Lag3 3.05 Aurkb 2.07 Wnt9a 8.48Wscd1 3.04 St6galnac6 2.06 Dusp14 8.43 Dkkl1 3.04 Mfge8 2.06 Dmd 8.29Zfp839 3.04 Slamf1 2.06 St6galnac2 8.26 Armcx1 3.03 Zdhhc2 2.06 Slc41a28.26 Zkscan16 3.03 Bag2 2.05 Cdh1 8.22 1700009P17Rik 3.02 Gpr55 2.05Tead1 8.09 Nkd1 3.01 Dip2a 2.05 Msc 8.05 Cdkn2b 3.01 BC064078 2.05Ttc39c 8.01 Igfbp6 3.01 S1pr5 2.05 Slc27a6 7.94 D1g2 3.01 Mlkl 2.04 Hlx7.85 Plscr1 3.00 Arap3 2.04 Mid2 7.73 Cacna1i 3.00 Ctla2a 2.04 Plod27.70 Ckm 3.00 Gypc 2.04 Prkcc 7.68 Kcnmb4 3.00 Ccdc88a 2.04 Ptgs1 7.63Arhgap8 2.99 Serpinb1a 2.03 6430571L13Rik 7.60 Gspt2 2.99 Fam19a3 2.03Specc1 7.55 Foxd2 2.98 Cdk1 2.03 Prss16 7.53 Vat1l 2.98 Trib1 2.01Fam129b 7.49 Bfsp2 2.98 Gadd45b 2.01 Spns3 7.49 Prrt2 2.98 Pfn2 2.01Lonrf3 7.45 Wbscr17 2.98 Klrd1 2.01 Fat4 7.41 Car13 2.98 Trp53i11 2.01Nphs2 7.39 Muc20 2.97 Cd22 2.00 Cpd 7.34 Bzrap1 2.96 Mtss1 2.00 Cdkn2a7.26 Frem2 2.96 Adam19 2.00 Il17rd 7.14 Fcer2a 2.96 Nusap1 2.00 Nebl7.12 Chn2 2.95 Hk3 0.50 Src 7.10 Blk 2.95 Pak1 0.50 Scnlb 7.07 Kcnk52.95 Irgc1 0.50 Sccpdh 7.04 Mpzl1 2.95 Zfp296 0.50 Igsf9b 7.03 Aplp12.95 Il6st 0.50 Crispld2 7.02 Tlr4 2.95 Oas2 0.49 Plscr4 7.01 H2-DMb22.95 Il6ra 0.49 Tmigd1 6.96 Lyn 2.94 Ctsf 0.49 Camk2n1 6.95 Meis2 2.94Lef1 0.49 Trat1 6.87 Camk1 2.93 Gm4951 0.49 Mtap6 6.85 Pacsin3 2.93Gpr133 0.49 Qpct 6.85 Tub 2.93 Olfr1033 0.49 Thsd7b 6.82 Cebpd 2.93Bambi-psi 0.48 Tceal3 6.76 Gm5111 2.92 Gtf2ird1 0.48 Nlrp12 6.75 Fasl2.92 Dtx1 0.48 2010002N04Rik 6.70 Lpcat2 2.91 Maml3 0.47 Lat2 6.68Mybpc2 2.90 Plaur 0.47 Susd2 6.61 Sytl3 2.90 Id3 0.47 Ptprj 6.61 H2-Eb22.90 Tnfrsf25 0.47 P2ry14 6.56 Hdac11 2.89 Susd4 0.47 Csgalnact1 6.55Slc43a3 2.89 Wfikkn2 0.47 Klri2 6.54 Klf12 2.89 4921525O09Rik 0.461700001C19Rik 6.48 Blnk 2.89 Klra3 0.46 Spp1 6.43 Atp2b4 2.881110032F04Rik 0.46 Hlf 6.41 Faim3 2.88 Car12 0.46 Gpx8 6.39 C1qc 2.88Pard6g 0.46 a 6.37 Epb4.1l3 2.87 Slc7a4 0.46 Rtn4r 6.31 Ms4a1 2.87 Lrp120.46 Lamc1 6.25 Prr5l 2.86 Spats2l 0.45 Kndc1 6.23 Fcrla 2.86 Dleu7 0.45Slc22a15 6.19 Ell3 2.86 Cacna2d4 0.45 Id2 6.15 Prdx4 2.85 Dst 0.45Tnfsf13b 6.14 Adcy9 2.85 Osbpl6 0.45 Elovl4 6.13 Pbx3 2.85 Tmem108 0.445830411N06Rik 6.04 Chmp4c 2.84 4930417O13Rik 0.44 Plxnd1 6.00 Osbpl1a2.84 Angptl2 0.44 Vstm2a 5.98 Apbb1 2.84 Adam6b 0.44 Rora 5.96 Cd79a2.84 Lrrc9 0.44 Il2 5.93 Slc6a8 2.83 2310042E22Rik 0.44 Klrc1 5.92Zcchc14 2.83 Lars2 0.44 Tesc 5.86 5730416F02Rik 2.83 Ankrd55 0.44 Cd40lg5.84 Gm9199 2.82 Ly6k 0.43 Glis2 5.84 Ern1 2.82 Kazn 0.43 Mt1 5.83 Lxn2.82 Dapl1 0.43 Mdfic 5.82 Armc2 2.82 Slc16a5 0.43 P2ry2 5.73 Cr2 2.82Unc13a 0.42 Hpse 5.72 Ptpn13 2.81 Gpr113 0.42 Ttc16 5.70 Bcl11a 2.81Siglech 0.42 Kitl 5.66 Scd1 2.81 Olig3 0.42 Arhgef25 5.65 Chst3 2.80Ksr2 0.42 Foxf2 5.62 Gcnt2 2.80 Card9 0.42 Kir3dl2 5.56 Lyl1 2.80 Rasip10.42 Unc79 5.52 Itga2 2.79 Dlc1 0.41 Aim1l 5.47 Scn4a 2.78 Gm19705 0.41Rgag4 5.44 Ccl5 2.78 Syde2 0.41 Pygl 5.41 March1 2.76 Actn1 0.41 Efcab65.39 Siglecg 2.76 Epx 0.41 Htra3 5.39 Islr 2.76 Mir5109 0.41 Card10 5.39Spib 2.76 Gpr83 0.40 Nek6 5.38 Ndrg1 2.75 Neb 0.40 6720401G13Rik 5.36Tex15 2.75 Sall2 0.40 Fam131b 5.36 Rgs1 2.74 Fam5b 0.40 Grb14 5.345031414D18Rik 2.74 Prickle1 0.40 Pde1c 5.33 Frmd4b 2.74 Nme4 0.40 Pld15.31 Ankrd6 2.74 Lif 0.40 Gipc2 5.30 Bcar3 2.74 Auts2 0.39 8430427H17Rik5.30 Rhbdfl 2.72 Col6a4 0.39 Gabarapl1 5.29 Slco4a1 2.72 Ccdc164 0.39Olfr527 5.28 Capg 2.71 Apol11b 0.39 Tenc1 5.27 H2-DMb1 2.71 Plac8 0.39Snx7 5.27 Hhex 2.71 Kcnf1 0.38 Rgs8 5.25 Postn 2.71 Gm15708 0.38 Slc16a75.24 Myole 2.71 Wnt5b 0.38 Alcam 5.23 Il15 2.70 Plat 0.38 Tmbim1 5.23Soat2 2.69 Nmnat2 0.38 Nefl 5.22 Casp1 2.69 4933440M02Rik 0.38 Pdcd15.21 Itga3 2.69 Dcaf12l1 0.38 2810030E01Rik 5.21 Rtn1 2.68 Nacc2 0.38Abhd3 5.19 Sema6b 2.68 She 0.37 Clnk 5.18 Ciita 2.68 Snord69 0.37 Nlrp1b5.14 Haao 2.68 Mical3 0.37 Cav2 5.13 Atf6 2.68 Cox6a2 0.36 Aff2 5.12Kcng1 2.67 Dmbt1 0.36 Kbtbd13 5.10 Ptk7 2.67 Aldh1l1 0.36 Ppap2a 5.07Kdelc2 2.67 Scarna6 0.36 Gm14718 5.06 Cd180 2.67 Lrp3 0.35 Pde4a 5.03Rhbdl3 2.66 Car11 0.35 Cnga2 4.95 Cd86 2.66 Il1r2 0.35 Acvrl1 4.92 Naip52.66 Atp6v1g3 0.35 Mapkapk3 4.92 Slc15a3 2.65 Clec9a 0.34 Ccr2 4.87BC013712 2.65 Slc6a9 0.34 Hip1 4.84 Cd74 2.64 Tnni3 0.34 Pkp2 4.83C77080 2.64 Gpr125 0.34 Nap1l3 4.83 Fam43 a 2.63 Ikzf2 0.34 Gorasp1 4.82S100a6 2.63 Slc7a10 0.33 Zfp651 4.82 Lmo2 2.63 Lman1l 0.33 Hmga2-ps14.81 Col8a2 2.63 A430105I19Rik 0.33 Tceal1 4.81 Celf5 2.63 Eng 0.32Atp1a2 4.81 Lhfpl4 2.63 Snora17 0.32 2200002K05Rik 4.81 Col1a1 2.62Gria4 0.32 Mmp2 4.77 Ppap2c 2.62 Fgfr1 0.32 Ankrd35 4.77 Ccl3 2.62BC048644 0.31 Klrc2 4.77 Rasgrp3 2.62 D830046C22Rik 0.31 Ptgfrn 4.74Klhl30 2.62 Hs6st2 0.31 Ildr1 4.66 Clu 2.62 Hoxa5 0.31 Ptpn3 4.64 Cd192.61 Alpl 0.30 5031425F14Rik 4.62 Syk 2.61 Pgpep1l 0.30 2510009E07Rik4.58 Cpne7 2.61 Adam6a 0.29 Emilin2 4.58 Rasgef1b 2.61 Hoxa3 0.292900026A02Rik 4.57 Gm11435 2.60 Fam110b 0.28 Cd101 4.54 Vpreb3 2.60Padi1 0.28 9030425E11Rik 4.52 Lhx2 2.58 Serpina9 0.28 1500009L16Rik 4.50Cybb 2.58 Igfbp4 0.28 Cdh17 4.48 Mapk8ip1 2.58 Pdzklip1 0.27 Clec5a 4.48Hck 2.57 Fzd1 0.27 Dnahc9 4.47 1110046J04Rik 2.57 4930546C10Rik 0.27Gpr25 4.45 Fam59a 2.57 Foxp3 0.26 Ifng 4.44 Stx11 2.56 Hoxa6 0.26 Socs24.42 Btk 2.56 Hoxa7 0.26 Pard3 4.42 H1f0 2.56 Edn3 0.26 Nhsl2 4.41 Vav32.56 Btc 0.26 Layn 4.40 F2rl2 2.55 Myl10 0.26 Csf2 4.35 Hspa2 2.55 Hoxa10.25 Crybb3 4.30 Slc29a4 2.55 Dnahc7a 0.24 Cxcr6 4.30 Tubb4a 2.54 Mira0.24 Zeb2 4.27 Arsb 2.54 Pcdhga10 0.24 Prkar2b 4.27 Trim7 2.54 Lrrc3b0.23 Fam70a 4.26 Mef2c 2.54 Slc4a4 0.23 Gdpd5 4.23 Asb14 2.54 Rprl3 0.23Gm8773 4.22 2010001M09Rik 2.54 Shc2 0.22 Fbn1 4.20 Ehd2 2.54 Expi 0.22Itgax 4.20 Itpripl2 2.53 Shisa2 0.22 Rbpj 4.17 Ifitm3 2.53 Gm12709 0.22Cxcl13 4.15 Zfp941 2.53 Fbln2 0.21 Tmem2 4.14 Pax5 2.53 Sec1 0.21Tmem205 4.14 Cd24a 2.52 C85492 0.21 Klrc3 4.13 Arhgef40 2.51 Lamc3 0.21Gcnt1 4.13 Rab30 2.51 Dbx1 0.20 Zc3h12c 4.12 H2-Eb1 2.50 Atp1b1 0.20Mir1199 4.09 Serpinb6a 2.50 Nek5 0.19 Ermn 4.08 Kynu 2.50 Actg2 0.19Agap1 4.07 Klra7 2.48 Dntt 0.17 1810033B17Rik 4.07 Maf 2.48 Nrg2 0.17Klf8 4.06 Fcgr2b 2.48 Sostdc1 0.14 4831426119Rik 4.06 H2-Ea-ps 2.47Trnp1 0.14 Ninl 4.06 Crip3 2.47 Lama1 0.14 Fbxo44 4.04 Abhd4 2.47 Rprl20.13 Rab39b 4.03 Fcrl1 2.47 0610012H03Rik 0.12 Tspan2 4.02 Egln3 2.47Lrrc32 0.10 Adrb1 4.00 Kif15 2.46 Spns2 3.98 H2-Ab1 2.46 Osbpl3 3.97Jazf1 2.46 Rapsn 3.96 Dennd5a 2.46 Dhrs9 3.94 Hgfac 2.45 Mboat2 3.90Rtp3 2.45 Pparg 3.90 9130206I24Rik 2.45 Clip4 3.89 Cited2 2.45 Apol9a3.88 Sult4a1 2.45 Marveld2 3.86 Gls2 2.44 Capn5 3.86 Cd80 2.44 Ranbp173.86 Tmem176b 2.44 Ncam1 3.85 Vcam1 2.44 Ctnnd2 3.85 Ptms 2.43 Slc25a243.85 Tmem154 2.43 Palm 3.84 Ccl4 2.43 Fcer1g 3.79 Rgs16 2.42 Gzmc 3.78Pawr 2.42 Prdm1 3.78 Wdfy4 2.42 Scn8a 3.75 Olfm1 2.42 Fkbp9 3.72 H2-Aa2.42 Gstt1 3.71 Stac2 2.41 Adssl1 3.71 Il18 2.40 Prrg4 3.71 Fam164a 2.40Kir3dl1 3.70 Phlda3 2.39 Ssbp2 3.67 Ctnnal1 2.39 Swap70 3.67 Tnfsf102.39 Trp73 3.67 Cubn 2.38 Il12rb2 3.66 Abi2 2.38 Phka1 3.662410004P03Rik 2.38 Xcr1 3.65 Trim36 2.38 Stau2 3.64 Bmf 2.379130019P16Rik 3.63 Adap1 2.36 Clec7a 3.63 Rarg 2.36

To evaluate the requirement of these ‘NK-type’ CD8 T cells in diseasepathogenesis, an adoptive transfer approach was used. CytotoxicCD8⁺NKG2D⁺ cells transferred alone, as well as total lymph node cells,were both able to give rise to AA in five recipients, whereas lymph nodepopulations depleted of NKG2D⁺ cells were unable to transfer disease(FIG. 54H). Not only are ‘NK-type’ CD8⁺NKG2D+ T cells the dominant celltype in the dermal infiltrate, but, moreover, they are both necessaryand sufficient for T cell mediated transfer of alopecia areata.

Using comparative genomics in order to characterize the transcriptionallandscape of AA lesional skin from C3H/HeJ mice as well as humans withAA, AFFYMETRIX™ profiling was first performed of whole skin from C3H/HeJmice with spontaneous AA versus unaffected C3H/HeJ skin (FIG. 55A, toppanel and FIG. 60). In parallel, human skin was similarly profiled fromperilesional biopsies of active disease in five AA patients versusnormal controls (Tables 15-17).

TABLE 15 Differentially expressed genes in the skin of alopecic femaleC3H/HeJ mice vs. skin obtained from age-matched female C3H/HeJ micewithout alopecia (n = 3 per group). Affy_ID GeneSymbol FC Affy_IDGeneSymbol FC 1419762_at Ubd 135.60 1455975_x_at Rnf114 1.76 1418652_atCxcl9 74.98 1453076_at Batf3 1.76 1417898_a_at Gzma 56.37 1419125_atPtpn18 1.76 1419697_at Cxcl11 53.33 1424524_at Dram1 1.76 1418930_atCxcl10 37.58 1453202_at E330016A19Rik 1.76 1419042_at Iigp1 34.651428346_at Trafd1 1.76 1418776_at Gbp8 33.06 1451987_at Arrb2 1.761418126_at Ccl5 27.95 1417790_at Dok1 1.76 1419043_a_at Iigp1 25.361418244_at Naa20 1.75 1438676_at Gbp6 25.11 1416226_at Arpc1b 1.751423467_at Ms4a4b 23.27 1438634_x_at Lasp1 1.75 1453196_a_at Oasl2 23.061419209_at Cxcl1 1.75 1419060_at Gzmb 21.74 1451363_a_at Dennd2d 1.751417141_at Igtp 21.08 1430622_at 4833423F13Rik 1.74 1435639_at2610528A11Rik 20.88 1447788_s_at Tspyl3 1.74 1423555_a_at Ifi44 20.581456310_a_at 2610002J02Rik 1.74 1417292_at Ifi47 18.63 1455251_at Itga11.74 1425394_at BC023105 18.43 1416750_at Sigmar1 1.74 1420549_at Gbp117.81 1421830_at Ak4 1.74 1417793_at Irgm2 17.25 1417346_at Pycard 1.741435906_x_at Gbp2 15.15 1436958_x_at Tpm3 1.74 1450033_a_at Stat1 14.481434069_at Prex1 1.74 1418825_at Irgm1 14.46 1440249_at Aknad1 1.741444078_at Cd8a 14.23 1442849_at Lrp1 1.74 1422812_at Cxcr6 13.981451174_at Lrrc33 1.73 1427747_a_at Lcn2 13.89 1451411_at Gprc5b 1.731420699_at Clec7a 13.82 1460469_at Tnfrsf9 1.73 1418240_at Gbp2 13.321452661_at Tfrc 1.73 1450783_at Ifit1 13.13 1430534_at Rnase6 1.731418392_a_at Gbp3 12.61 1456028_x_at Marcks 1.73 1424305_at Igj 12.531448731_at Il10ra 1.73 1434380_at Gbp7 12.51 1418257_at Slc12a7 1.731424865_at Pyy 12.17 1420170_at Myh9 1.72 1422588_at Krt6b 12.071424032_at Hvcn1 1.72 1434046_at AA467197 11.98 1435748_at Gda 1.721418580_at Rtp4 11.29 1451285_at Fus 1.72 1419709_at Stfa3 11.041454607_s_at Psat1 1.72 1425156_at Gbp7 10.72 1434089_at Synpo 1.711449254_at Spp1 10.61 1421525_a_at Naip5 1.71 1440481_at Stat1 10.531458415_at Clec2e 1.71 1449153_at Mmp12 10.18 1419721_at Niacr1 1.711424921_at Bst2 9.80 1453328_at 2700008G24Rik 1.71 1429947_a_at Zbp19.65 1435337_at Tshz3 1.71 1456907_at Cxcl9 9.62 1456011_x_at Acaa1a1.71 1421075_s_at Cyp7b1 8.74 1423986_a_at Shisa5 1.71 1451777_at Ddx608.72 1427386_at Arhgef16 1.70 1421688_a_at Ccl1 8.65 1443123_at Tanc21.70 1421074_at Cyp7b1 8.56 1418674_at Osmr 1.70 1419604_at Zbp1 8.511445588_at Ankrd44 1.70 1419714_at Cd274 8.41 1424552_at Casp8 1.701427102_at Slfn4 8.40 1439261_x_at Mitd1 1.70 1424761_at Fam115c 8.261452428_a_at B2m 1.70 1419135_at Ltb 8.24 1418045_at Inpp1 1.701450034_at Stat1 7.98 1441317_x_at Jakmip1 1.70 1425832_a_at Cxcr6 7.981436589_x_at Prkd2 1.70 1420915_at Stat1 7.67 1437503_a_at Shisa5 1.701438037_at Herc6 7.62 1421578_at Ccl4 1.70 1448436_a_at Irf1 7.611435227_at Bcl11b 1.70 1448932_at Krt16 7.12 1436177_at Plekha2 1.701435710_at AI661384 7.10 1457644_s_at Cxcl1 1.70 1451564_at Parp14 7.081429527_a_at Plscrl1 1.70 1452614_at Bcl2l15 7.00 1433531_at Acsl4 1.691417256_at Mmp13 6.88 1420731_a_at Csrp2 1.69 1447541_s_at Itgae 6.791438097_at Rab20 1.69 1432026_a_at Herc6 6.70 1426239_s_at Arrb2 1.691434372_at AW112010 6.62 1427182_s_at D18Ertd653e 1.69 1459913_atTnfsf10 6.51 1435338_at Cdk6 1.69 1451426_at Dhx58 6.51 1433885_atIqgap2 1.69 1452405_x_at Trav9d-3 6.44 1450852_s_at F2r 1.69 1429570_atMlkl 6.41 1446507_at Kif24 1.69 1444064_at Samhd1 6.28 1450456_at Il21r1.69 1451860_a_at Trim30a 6.23 1418612_at Slfn1 1.68 1453080_at Apol7a6.22 1438974_x_at Pitpnm1 1.68 1420380_at Ccl2 6.12 1439030_at Gmppb1.68 1434438_at Samhd1 6.11 1416619_at 4632428N05Rik 1.68 1450696_atPsmb9 6.06 1441880_x_at Tmem149 1.68 1451537_at Chi3l1 5.99 1448797_atElk3 1.68 1450165_at Slfn2 5.88 1443794_x_at Noc4l 1.68 1424727_at Ccr55.83 1452289_a_at Rnf135 1.68 1421256_at Gzmc 5.80 1450918_s_at Src 1.671448162_at Vcam1 5.80 1420955_at Vsnl1 1.67 1453913_a_at Tap2 5.791418751_at Sit1 1.67 1418191_at Usp18 5.73 1447568_at Gatc 1.671425005_at Klrc1 5.65 1460700_at Stat3 1.67 1439680_at Tnfsf10 5.641419911_at Coro1c 1.66 1433935_at AU020206 5.51 1452966_at Bcl11b 1.661426113_x_at Trav9d-3 5.51 1421550_a_at Trim34a 1.66 1422415_at Ang25.44 1423686_a_at Prr13 1.66 1460245_at Klrd1 5.41 1455287_at Cdk6 1.661426971_at Uba7 5.34 1439422_a_at Fam132a 1.66 1443698_at Xaf1 5.321416935_at Trpv2 1.66 1418131_at Samhd1 5.26 1436183_at Zc3hav1 1.661419591_at Gsdmc 5.20 1451314_a_at Vcam1 1.66 1416897_at Parp9 5.111417300_at Smpdl3b 1.66 1417244_a_at Irf7 5.08 1419097_a_at Stom 1.661449216_at Itgae 5.06 1446939_at Trim12a 1.65 1448632_at Psmb10 5.061423411_at Rbm47 1.65 1439825_at Dtx3l 5.02 1450355_a_at Capg 1.651416714_at Irf8 5.00 1436220_at Zfp287 1.65 1425917_at H28 4.991429742_at Rcbtb2 1.65 1447621_s_at Tmem173 4.93 1455221_at Abcg1 1.651425396_a_at Lck 4.88 1438934_x_at Sema4a 1.65 1418536_at H2-Q7 4.841452836_at Lpin2 1.65 1449025_at Ifit3 4.75 1427874_at Rnf114 1.641436649_at Ikzf3 4.75 1452425_at Tnfrsf14 1.64 1425947_at Ifng 4.711422303_a_at Tnfrsf18 1.64 1417172_at Ube2l6 4.70 1418396_at Gpsm3 1.641438855_x_at Tnfaip2 4.70 1451462_a_at Ifnar2 1.64 1422962_a_at Psmb84.69 1447711_x_at 4933412E12Rik 1.64 1421186_at Ccr2 4.66 1454757_s_atIfi27l1 1.64 1418204_s_at Aif1 4.61 1424246_a_at Tes 1.64 1421911_atStat2 4.57 1459872_x_at H2-DMa 1.64 1450753_at Nkg7 4.55 1424304_atTpcn2 1.64 1450424_a_at Il18bp 4.53 1416695_at Tspo 1.64 1450403_atStat2 4.52 1451475_at Plxnd1 1.64 1452565_x_at LOC641050 4.44 1428727_atCep192 1.64 1441054_at Apol8 4.43 1440779_s_at Slc5a9 1.63 1449328_atLy75 4.39 1433451_at Cdk5r1 1.63 1422601_at Serpinb9 4.39 1450531_atH2-Bl 1.63 1422177_at Il13ra2 4.39 1458601_at 8030447M02Rik 1.631426170_a_at Cd8b1 4.38 1426739_at Donson 1.63 1460603_at Samd9l 4.371423350_at Socs5 1.63 1448754_at Rbp1 4.31 1456467_s_at Nlk 1.631417822_at D17H6S56E-5 4.28 1447803_x_at Capg 1.63 1435208_at Dtx3l 4.261453201_at Rassf10 1.63 1418649_at Egln3 4.26 1438148_at Cxcl3 1.631422160_at H2-T24 4.23 1444197_at Rin1 1.63 1456064_at AI504432 4.211428859_at Paox 1.63 1449184_at Pglyrp1 4.19 1455656_at Btla 1.631438498_at Zmynd15 4.19 1455080_at Ppplr16b 1.63 1449195_s_at Cxcl164.11 1424857_a_at Trim34a 1.62 1439034_at Spn 4.10 1450140_a_at Cdkn2a1.62 1450678_at Itgb2 4.10 1438095_x_at Noc4l 1.62 1437176_at Nlrc5 4.091460231_at Irf5 1.62 1458299_s_at Nfkbie 4.07 1426318_at Serpinb1b 1.621421262_at Lipg 4.06 1427348_at Zc3h12a 1.62 1426039_a_at Alox12e 4.051451862_a_at Prf1 1.62 1429184_at Gvin1 4.05 1442944_at C76555 1.621448380_at Lgals3bp 4.04 1420635_a_at Tcirg1 1.62 1436576_at Fam26f 4.011435143_at Elk3 1.62 1453939_x_at Gm9706 3.99 1449623_at Txnrd3 1.611434457_at Sp100 3.98 1458345_s_at Colec11 1.61 1425295_at Ear11 3.971423812_s_at Vopp1 1.61 1425065_at Oas2 3.97 1417561_at Apoc1 1.611449846_at Ear2 3.97 1455818_at 4930427A07Rik 1.60 1450582_at H2-Q5 3.961426757_at Ampd2 1.60 1440926_at Flt1 3.94 1418265_s_at Irf2 1.601457140_s_at Rassf10 3.91 1434185_at Acaca 1.60 1455500_at Rnf213 3.901437327_x_at Enoph1 1.60 1426970_a_at Uba7 3.89 1456240_x_at Cdca4 1.601448452_at Irf8 3.88 1443086_at Alcam 1.60 1451673_at Cd8a 3.881442018_at Btg1 1.60 1437929_at Dact2 3.86 1418686_at Oas1c 1.601426774_at Parp12 3.84 1441843_s_at 5230400M03Rik 1.60 1418648_at Egln33.82 1443882_at Slc26a2 1.60 1437811_x_at Cotl1 3.78 1420909_at Vegfa1.60 1428420_a_at 1200009I06Rik 3.78 1439587_at Lemd3 1.60 1417961_a_atTrim30a 3.77 1433169_at 5830456J23Rik 1.60 1425225_at Fcgr4 3.741437451_at Ecscr 1.59 1421812_at Tapbp 3.74 1436074_at Nfkbid 1.591451905_a_at Mx1 3.72 1437789_at Birc6 1.59 1441752_at Art3 3.691450716_at Adamts1 1.59 1448301_s_at Serpinb1a 3.69 1438828_at Rapgef61.59 1436172_at Gm20559 3.68 1453064_at Etaa1 1.59 1418133_at Bcl3 3.671439047_s_at Recql 1.59 1418655_at B4galnt1 3.66 1435316_at Psma6 1.591417821_at D17H6S56E-5 3.66 1428484_at Osbpl3 1.59 1417171_at Itk 3.631417627_a_at Limk1 1.59 1460227_at Timp1 3.61 1459884_at Cox7c 1.591455161_at AI504432 3.61 1455660_at Csf2rb 1.59 1421228_at Ccl7 3.601448511_at Ptprcap 1.59 1434905_at Ndufa4l2 3.60 1440245_at Ccdc88b 1.591460218_at Cd52 3.60 1418587_at Traf3 1.59 1460437_at Cyth4 3.541424339_at Oasl1 1.58 1430005_a_at Batf2 3.53 1416166_a_at Prdx4 1.581450188_s_at Lipg 3.53 1427095_at Cdcp1 1.58 1419698_at Cxcl11 3.521456288_at Slfn5 1.58 1439790_at Serpinb9 3.51 1452239_at Gt(ROSA)26Sor1.58 1419413_at Ccl17 3.51 1449516_a_at Rgs3 1.58 1421009_at Rsad2 3.501424829_at A830007P12Rik 1.58 1436838_x_at Cotl1 3.50 1454402_at Zfp9421.58 1455269_a_at Coro1a 3.50 1434139_at Parp11 1.58 1422828_at Cd3d3.49 1428583_at Nufip2 1.58 1440866_at Eif2ak2 3.47 1451097_at Vasp 1.581420437_at Ido1 3.45 1458595_at Intu 1.58 1439814_at Atp8b4 3.441457550_at 9530059O14Rik 1.58 1441706_at Dscaml1 3.44 1419530_at Il12b1.58 1421322_a_at Irf9 3.43 1447762_x_at Car12 1.58 1427076_at Mpeg13.43 1417588_at Galnt3 1.58 1435454_a_at BC006779 3.41 1437052_s_atSlc2a3 1.58 1444619_x_at Psmb8 3.40 1435084_at C730049O14Rik 1.581444350_at Slfn10-ps 3.39 1419235_s_at Helb 1.57 1425335_at Cd8a 3.381440147_at Lgi2 1.57 1450484_a_at Cmpk2 3.38 1460347_at Krt14 1.571435832_at Lrrc4 3.38 1452991_at Chd2 1.57 1424067_at Icam1 3.371429758_at 1700017B05Rik 1.57 1416318_at Serpinb1a 3.37 1449508_atIl27ra 1.57 1445897_s_at Ifi35 3.35 1416087_at Ap1s1 1.57 1450291_s_atMs4a4c 3.34 1438673_at Slc4a7 1.57 1416273_at Tnfaip2 3.34 1438750_atAtrx 1.57 1453304_s_at Ly6e 3.33 1423869_s_at Txnrd3 1.57 1418770_at Cd23.32 1420692_at Il2ra 1.57 1434067_at AI662270 3.32 1449227_at Ch25h1.57 1428735_at Cd69 3.31 1425241_a_at Wsb1 1.57 1424965_at Lpxn 3.311450387_s_at Ak4 1.56 1455393_at Cp 3.31 1427164_at Il13ra1 1.561429567_at Rassf10 3.30 1455084_x_at Shmt2 1.56 1436905_x_at Laptm5 3.301452169_a_at Dgkz 1.56 1428485_at Car12 3.28 1420505_a_at Stxbp1 1.561456211_at Nlrp10 3.28 1455839_at Uggt1 1.56 1438531_at A730054J21Rik3.26 1428118_at Lingo1 1.56 1449175_at Gpr65 3.25 1427539_a_at Zwint1.56 1424923_at Serpina3g 3.24 1418271_at Bhlhe22 1.56 1417995_at Ptpn223.23 1447851_x_at Atp10a 1.56 1440990_at Kif26b 3.20 1452837_at Lpin21.56 1420338_at Alox15 3.18 1420171_s_at Myh9 1.55 1419300_at Flt1 3.171439253_x_at Prelid1 1.55 1419128_at Itgax 3.16 1452803_at Glipr2 1.551426872_at Fcgbp 3.15 1456914_at Slc16a4 1.55 1425336_x_at H2-K1 3.151448202_x_at Prelid1 1.55 1448759_at Il2rb 3.14 1452391_at Cxadr 1.551419178_at Cd3g 3.11 1435595_at 1810011O10Rik 1.55 1420412_at Tnfsf103.11 1452903_at 6230427J02Rik 1.55 1433465_a_at AI467606 3.11 1451110_atEgln1 1.55 1418203_at Pmaip1 3.11 1435564_at Neurl1b 1.55 1430208_atKrt42 3.09 1422489_at Mogs 1.55 1421628_at Il18r1 3.08 1452337_at4930427A07Rik 1.54 1417620_at Rac2 3.08 1459983_at Impa2 1.54 1454850_atTbc1d10c 3.07 1432997_at 5830462P14Rik 1.54 1439221_s_at Cd40 3.071457304_at D13Ertd787e 1.54 1438658_a_at S1pr3 3.06 1417955_at Ccdc711.54 1426278_at Ifi27l2a 3.06 1428034_a_at Tnfrsf9 1.54 1429692_s_atGch1 3.04 1439455_x_at Capza1 1.54 1458148_at Nlrc3 3.03 1451608_a_atTspan33 1.54 1440927_x_at Apol11b 3.03 1417878_at E2f1 1.54 1436236_x_atCotl1 3.03 1448894_at Akr1b8 1.54 1448940_at Trim21 3.02 1455067_at Psd41.53 1421217_a_at Lgals9 3.02 1457410_at Arhgap5 1.53 1451756_at Flt13.01 1436848_x_at Impa1 1.53 1455048_at Igsf3 3.01 1436395_at Card6 1.531435560_at Itgal 3.01 1436729_at Afap1 1.53 1425719_a_at Nmi 2.991437185_s_at Tmsb10 1.53 1439956_at Ms4a6b 2.98 1428018_a_at AF2517051.53 1436562_at Ddx58 2.98 1431528_at 5830427D02Rik 1.53 1422632_at Ctsw2.98 1425760_a_at Pitpnm1 1.53 1436472_at Slfn9 2.98 1418492_at Grem21.53 1427013_at Car9 2.98 1445201_at Zfp53 1.53 1449361_at Tbx21 2.981459894_at Iqgap2 1.53 1426025_s_at Laptm5 2.97 1438154_x_at2610002J02Rik 1.53 1438052_at A130071D04Rik 2.97 1434573_at Traf3ip31.53 1426276_at Ifih1 2.97 1433739_at Nol10 1.53 1456890_at Ddx58 2.961434070_at Jag1 1.52 1416295_a_at Il2rg 2.94 1423826_at Noc4l 1.521434873_a_at Acap1 2.93 1416875_at Parvg 1.52 1456103_at Pml 2.931447792_x_at Gpr174 1.52 1418678_at Has2 2.92 1417128_at Plekho1 1.521420697_at Slc15a3 2.91 1427260_a_at Tpm3 1.52 1436058_at Rsad2 2.901416379_at Panx1 1.52 1425294_at Slamf8 2.90 1437239_x_at Phc2 1.521459151_x_at Ifi35 2.90 1451415_at 1810011O10Rik 1.52 1418991_at Bak12.89 1448713_at Stat4 1.52 1439068_at Erap1 2.89 1421937_at Dapp1 1.511424617_at Ifi35 2.89 1433748_at Zdhhc18 1.51 1417189_at Psme2 2.891454350_at Intu 1.51 1451544_at Tapbpl 2.88 1429128_x_at Nfkb2 1.511419412_at Xcl1 2.88 1418004_a_at Tmem176b 1.51 1419539_at Irx4 2.881423154_at BC005537 1.51 1452592_at Mgst2 2.87 1426677_at Flna 1.511420774_a_at 4930583H14Rik 2.86 1448600_s_at Vav3 1.51 1440169_x_atIfnar2 2.86 1457539_at D10Ertd709e 1.51 1420499_at Gch1 2.85 1420914_atSlco2a1 1.51 1416002_x_at Cotl1 2.85 1429247_at Anxa6 1.51 1423754_atIfitm3 2.84 1452411_at Lrrc1 1.51 1416942_at Erap1 2.84 1449619_s_atArhgap9 1.51 1449169_at Has2 2.83 1420351_at Tnfrsf4 1.51 1419609_atCcr1 2.83 1437242_at Ttll12 1.51 1449591_at Casp4 2.81 1439444_x_atTmed10 1.50 1436598_at Icos 2.81 1448933_at Pcdhb17 1.50 1427381_at Irg12.81 1434219_at Stim2 1.50 1419879_s_at Trim25 2.81 1455428_at Fam53b1.50 1449143_at Rtp4 2.80 1419226_at Cd96 1.50 1449839_at Casp3 2.781435169_at A930001N09Rik 0.67 1426599_a_at Slc2a1 2.78 1451348_at Deptor0.67 1452117_a_at Fyb 2.77 1425411_at Arl4a 0.67 1434773_a_at Slc2a12.77 1428864_at Dusp8 0.67 1450378_at Tapbp 2.76 1426522_at Hadhb 0.671419561_at Ccl3 2.76 1417312_at Dkk3 0.67 1427007_at Sash3 2.761418190_at Pon1 0.67 1422706_at Pmepa1 2.76 1454078_a_at Gal3st1 0.661427892_at Myo1g 2.75 1425145_at Il1rl1 0.66 1451335_at Plac8 2.731417421_at S100a1 0.66 1425974_a_at Trim25 2.72 1460192_at Osbpl1a 0.661426600_at Slc2a1 2.67 1419963_at Deptor 0.66 1418826_at Ms4a6b 2.671415743_at Hdac5 0.66 1430244_at 4921509J17Rik 2.66 1435559_at Myo6 0.661457664_x_at C2 2.65 1450395_at Slc22a5 0.66 1416564_at Sox7 2.651428469_a_at Dzip1 0.66 1433466_at AI467606 2.65 1429884_at Srgap2 0.661425801_x_at Cotl1 2.64 1450971_at Gadd45b 0.66 1435945_a_at Kcnn4 2.631436344_at C2cd2 0.66 1434268_at Adar 2.63 1418921_at Cadm3 0.651420353_at Lta 2.63 1421471_at Npy1r 0.65 1418741_at Itgb7 2.621453571_at Deptor 0.65 1416246_a_at Coro1a 2.62 1452283_at Rassf8 0.651422280_at Gzmk 2.61 1416268_at Ets2 0.65 1421596_s_at H28 2.601423364_a_at Aktip 0.65 1415834_at Dusp6 2.60 1453069_at Pik3cb 0.651438364_x_at Ang4 2.60 1422838_at Kcnu1 0.65 1439819_at AU015263 2.601433977_at Hs3st3b1 0.65 1451755_a_at Apobec1 2.59 1426010_a_at Epb4.1l30.65 1416296_at Il2rg 2.59 1455029_at Kif21a 0.65 1439276_at Adar 2.591460406_at Pls1 0.65 1417495_x_at Cp 2.59 1434581_at 2410066E13Rik 0.651453352_at Atp10b 2.58 1422740_at Tnfrsf21 0.65 1437478_s_at Efhd2 2.581435396_at Stxbp6 0.65 1434366_x_at C1qb 2.58 1419688_at Gpc6 0.651417185_at Ly6a 2.58 1418658_at Fam82b 0.65 1443858_at Trim12c 2.581419457_at Rgnef 0.65 1417494_a_at Cp 2.58 1443904_at Fads6 0.651427746_x_at H2-K1 2.57 1425186_at Lmbrd1 0.65 1421034_a_at Il4ra 2.561450286_at Npr3 0.65 1422005_at Eif2ak2 2.56 1425037_at Fgd4 0.641455000_at Gpr68 2.56 1437871_at Pgm5 0.64 1415803_at Cx3cl1 2.551416774_at Wee1 0.64 1422782_s_at Tlr3 2.55 1458129_at Rora 0.641416097_at Lrrc4 2.54 1433460_at Ttc7b 0.64 1460651_at Lat 2.531451200_at Kif1b 0.64 1427325_s_at Akna 2.53 1448320_at Stim1 0.641425603_at Tmem176a 2.49 1452309_at Cgnl1 0.64 1439595_at Tcra 2.491454824_s_at Mtus1 0.64 1424041_s_at C1s 2.49 1415981_at Herpud2 0.641418718_at Cxcl16 2.48 1419505_a_at Ggps1 0.64 1456061_at Gimap8 2.471457686_at Akap17b 0.63 1417056_at Psme1 2.46 1437149_at Slc6a6 0.631418981_at Casp12 2.46 1437661_at AU021092 0.63 1421211_a_at Ciita 2.461443906_at Cd55 0.63 1449235_at Fasl 2.45 1451714_a_at Map2k3 0.631449265_at Casp1 2.44 1435693_at Mall 0.63 1424383_at Tmem51 2.441451563_at Emr4 0.63 1425405_a_at Adar 2.43 1452333_at Smarca2 0.631425902_a_at Nfkb2 2.42 1428835_at Myh14 0.63 1452817_at Smyd3 2.421433711_s_at Sesn1 0.63 1416871_at Adam8 2.42 1448669_at Dkk3 0.631457917_at Lck 2.42 1416778_at Sdpr 0.63 1449127_at Selplg 2.421449082_at Mfap5 0.63 1416514_a_at Fscn1 2.42 1425891_a_at Grtp1 0.631439773_at Ly6e 2.41 1418250_at Arl4d 0.63 1436223_at Itgb8 2.411458882_at Serpinb8 0.62 1454268_a_at Cyba 2.41 1418714_at Dusp8 0.621451821_a_at Sp100 2.41 1431216_s_at Dnajc6 0.62 1444242_at Slco2a1 2.401426208_x_at Plagl1 0.62 1415983_at Lcp1 2.40 1425138_at Guca1b 0.621418293_at Ifit2 2.39 1425318_a_at Tmem116 0.62 1420671_x_at Ms4a4c 2.391423176_at Tob1 0.62 1450534_x_at H2-K1 2.39 1425515_at Pik3r1 0.621418077_at Trim21 2.39 1455665_at Lonrf1 0.62 1417496_at Cp 2.381426576_at Sgms1 0.62 1435415_x_at Marcksl1 2.37 1440559_at Hmga2-ps10.62 1451320_at Arhgap8 2.37 1417545_at Trpv4 0.62 1427041_at BC0137122.36 1418954_at Camkk1 0.62 1422139_at Plau 2.36 1424701_at Pcdh20 0.621441768_at 9430051O21Rik 2.35 1454881_s_at Upk3b 0.62 1449455_at Hck2.35 1419378_a_at Fxyd2 0.62 1421358_at H2-M3 2.34 1448926_at Hoxa5 0.621436312_at Ikzf1 2.34 1437731_at C2cd2 0.62 1444632_at BC064078 2.331436501_at Mtus1 0.61 1450791_at Nppb 2.33 1450110_at Adh7 0.611424927_at Glipr1 2.33 1417013_at Hspb8 0.61 1419764_at Chi3l3 2.331443969_at Irs2 0.61 1426093_at Trim34a 2.33 1450206_at Dlc1 0.611420515_a_at Pglyrp2 2.33 1428774_at Gpc6 0.61 1448061_at Msr1 2.331422904_at Fmo2 0.61 1418641_at Lcp2 2.32 1425321_a_at Clmn 0.611418842_at Hcls1 2.31 1426743_at Appl2 0.61 1434400_at Tgif2 2.311427945_at Dpyd 0.61 1439494_at Slc5a9 2.30 1428321_at Eml1 0.601448757_at Pml 2.28 1431226_a_at Fndc4 0.60 1431339_a_at Efhd2 2.281421848_at Slc22a5 0.60 1455581_x_at Gm20559 2.28 1428731_at Usp54 0.601437132_x_at Nedd9 2.27 1456939_at Fam154b 0.60 1456307_s_at Adcy7 2.271424408_at Lims2 0.60 1422932_a_at Vav1 2.27 1426139_a_at Ccrl1 0.601450446_a_at Socs1 2.27 1426332_a_at Cldn3 0.60 1434068_s_at AI6622702.26 1416823_a_at Osbplla 0.60 1416069_at Pfkp 2.26 1423994_at Kif1b0.60 1421210_at Ciita 2.26 1415904_at Lpl 0.60 1437785_at Adamts9 2.251429656_at Rhobtb1 0.60 1436230_at Gpr114 2.25 1421158_at Cgnl1 0.601425119_at Oas1b 2.25 1456150_at Jhdm1d 0.60 1449310_at Ptger2 2.251454604_s_at Tspan12 0.59 1440721_at 5930433N17Rik 2.25 1434307_atTmem64 0.59 1430352_at Adamts9 2.25 1431188_a_at Tom1 0.59 1456377_x_atLimd2 2.25 1416926_at Trp53inp1 0.59 1449991_at Cd244 2.25 1423447_atClpx 0.59 1416051_at C2 2.25 1451782_a_at Slc29a1 0.59 1455132_at Rasal32.24 1423174_a_at Pard6b 0.59 1422124_a_at Ptprc 2.24 1436293_x_at Ildr20.59 1448576_at Il7r 2.23 1444073_at Maf 0.59 1421931_at Icos 2.221447462_at D7Wsu130e 0.59 1436199_at Trim14 2.21 1424280_at Mospd1 0.591446509_at Smox 2.20 1451270_at Dusp18 0.59 1430148_at Rab19 2.201451207_at Micu1 0.59 1450170_x_at H2-D1 2.19 1455056_at Lmo7 0.591439736_at 5830453J16Rik 2.19 1437409_s_at Gpr126 0.59 1451567_a_atIfi203 2.19 1436988_at Ism1 0.58 1433920_at Sema4c 2.18 1420718_at Odz20.58 1420913_at Slco2a1 2.18 1425456_a_at Map2k3 0.58 1439148_a_at Pfkl2.18 1450318_a_at P2ry2 0.58 1452544_x_at H2-D1 2.18 1436853_a_at Snca0.58 1441600_at C920021A13 2.17 1424437_s_at Abcg4 0.58 1422804_atSerpinb6b 2.16 1426285_at Lama2 0.58 1453228_at Stx11 2.15 1417962_s_atGhr 0.58 1427682_a_at Egr2 2.15 1455340_at Dennd5b 0.58 1428378_atZc3hav1 2.15 1415874_at Spry1 0.58 1430165_at Stk17b 2.15 1435105_atRnf208 0.58 1451321_a_at Rbm43 2.15 1428240_at Nrxn1 0.58 1457780_atStx11 2.15 1425906_a_at Sema3e 0.58 1439764_s_at Igf2bp2 2.14 1454926_atSphkap 0.58 1435913_at B4galnt4 2.14 1416316_at Slc27a2 0.58 1449220_atGimap3 2.13 1434917_at Cobl 0.57 1456251_x_at Tspo 2.13 1429778_at Optn0.57 1422781_at Tlr3 2.13 1436350_at Fam171b 0.57 1423135_at Thy1 2.131452207_at Cited2 0.57 1424375_s_at Gimap4 2.12 1428547_at Nt5e 0.571428660_s_at Tor3a 2.12 1426263_at Cadm4 0.57 1439012_a_at Dck 2.121429036_at Otop3 0.57 1441912_x_at C2 2.12 1435763_at Tbc1d16 0.571424704_at Runx2 2.11 1420942_s_at Rgs5 0.57 1424953_at BC021614 2.111450974_at Timp4 0.57 1431843_a_at Nfkbie 2.10 1434639_at Klhl29 0.561417219_s_at Tmsb10 2.10 1421346_a_at Slc6a6 0.56 1448160_at Lcp1 2.101433742_at Kank1 0.56 1427352_at Krt79 2.10 1435207_at Dixdc1 0.561419194_s_at Gmfg 2.09 1450838_x_at Rpl37 0.56 1460675_at Igsf8 2.091457671_at Homer2 0.56 1426568_at Slc2a9 2.08 1422008_a_at Aqp3 0.561424961_at Tapbpl 2.08 1415864_at Bpgm 0.56 1418099_at Tnfrsf1b 2.081437181_at Peli2 0.56 1422527_at H2-DMa 2.08 1416072_at Cd34 0.561426554_a_at Pgam1 2.08 1460514_s_at Ascl2 0.56 1438941_x_at Ampd2 2.081420941_at Rgs5 0.56 1450648_s_at H2-Ab1 2.07 1423966_at Cd99l2 0.551440165_at Ptprc 2.07 1435435_at Cttnbp2 0.55 1437724_x_at Pitpnm1 2.071428336_at Agpat4 0.55 1434364_at Map3k14 2.06 1419458_at Rgnef 0.551419537_at Tfec 2.06 1424567_at Tspan2 0.55 1425728_at Gm12185 2.061419491_at Defb1 0.55 1455966_s_at Nudt21 2.05 1415865_s_at Bpgm 0.551419282_at Ccl12 2.05 1422667_at Krt15 0.55 1420404_at Cd86 2.051417355_at Peg3 0.55 1415765_at Hnrnpul2 2.05 1439478_at Acot2 0.551422511_a_at Ogfr 2.05 1450460_at Aqp3 0.55 1425548_a_at Lst1 2.051426510_at Sccpdh 0.55 1427683_at Egr2 2.05 1422996_at Acot2 0.551453757_at Herc6 2.04 1433454_at Abtb2 0.55 1435331_at Pyhin1 2.041436640_x_at Agpat4 0.55 1419119_at Hcst 2.03 1453008_at Trnp1 0.551458229_at Robo2 2.03 1424392_at Adhfe1 0.54 1422808_s_at Dock2 2.031417332_at Rfx2 0.54 1437213_at Nudt21 2.02 1419197_x_at Hamp 0.541429831_at Pik3ap1 2.01 1429001_at Pir 0.54 1439808_at Ipcef1 2.011438496_a_at Ddx26b 0.54 1428891_at Parm1 2.01 1454699_at Sesn1 0.541427765_a_at Tcrb-J 2.01 1437217_at Ankrd6 0.54 1426133_a_at Mitd1 2.001418412_at Tpd52l1 0.54 1425016_at Ephb2 2.00 1424747_at Kif1c 0.541422562_at Rrad 2.00 1454646_at Tcp11l2 0.54 1418110_a_at Inpp5d 2.001421040_a_at Gsta2 0.53 1420272_at Samhd1 2.00 1434326_x_at Coro2b 0.531417928_at Pdlim4 2.00 1417027_at Trim2 0.53 1460220_a_at Csf1 2.001460242_at Cd55 0.53 1451828_a_at Acsl4 2.00 1453119_at Otud1 0.531435672_at 3830612M24 1.99 1420688_a_at Sgce 0.53 1453181_x_at Plscr11.99 1450119_at Dst 0.53 1428767_at Gsdmd 1.99 1448119_at Bpgm 0.531436861_at Il7 1.99 1456481_at Esyt3 0.53 1437270_a_at Clcf1 1.991422168_a_at Bdnf 0.53 1419202_at Cst7 1.98 1434542_at Gpt2 0.521435529_at Gm14446 1.98 1422905_s_at Fmo2 0.52 1419810_x_at Arhgap9 1.981425809_at Fabp4 0.52 1429525_s_at Myo1f 1.98 1439549_at Prrg3 0.521437308_s_at F2r 1.98 1448416_at Mgp 0.52 1419676_at Mx2 1.98 1437197_atSorbs2 0.52 1455019_x_at Ckap4 1.98 1422619_at Ppap2a 0.52 1438948_x_atTspo 1.97 1436737_a_at Sorbs1 0.52 1448618_at Mvp 1.97 1422620_s_atPpap2a 0.52 1436365_at Zbtb7c 1.97 1427378_at Krt75 0.52 1418464_atMatn4 1.97 1416779_at Sdpr 0.51 1439093_at Hspa4l 1.97 1451501_a_at Ghr0.51 1435193_at A230050P20Rik 1.97 1423506_a_at Nnat 0.51 1439065_x_atGm13152 1.97 1422974_at Nt5e 0.51 1423306_at 2010002N04Rik 1.971425761_a_at Nfatc1 0.51 1430658_a_at Gsdma2 1.97 1458268_s_at Igfbp30.51 1419469_at Gnb4 1.96 1460241_a_at St3gal5 0.50 1426725_s_at Ets11.96 1417028_a_at Trim2 0.50 1417288_at Plekha2 1.96 1456397_at Cdh40.50 1456586_x_at Mvp 1.96 1434156_at Rab11fip4 0.50 1426415_a_at Trim251.95 1429024_at Rbm20 0.50 1437249_at Skap1 1.95 1441254_at Pard3b 0.501433517_at Myeov2 1.95 1419289_a_at Syngr1 0.50 1425374_at Oas3 1.951422112_at Ccbp2 0.50 1425681_a_at Pmd 1.95 1444559_at Phtf2 0.501436659_at Dclk1 1.94 1451410_a_at Crip3 0.50 1446326_at Col1a2 1.941420017_at Tspan8 0.50 1435596_at Pion 1.94 1416194_at Cyp4b1 0.501416492_at Ccne1 1.94 1434013_at Ablim3 0.49 1422701_at Zap70 1.931419706_a_at Akap12 0.49 1435517_x_at Ralb 1.93 1451762_a_at Kif1b 0.491436902_x_at Tmsb10 1.93 1427371_at Abca8a 0.49 1425166_at Rbl1 1.931424007_at Gdf10 0.49 1421207_at Lif 1.93 1424155_at Fabp4 0.481460134_at 9330175E14Rik 1.93 1428357_at 2610019F03Rik 0.48 1420273_x_atSamhd1 1.93 1454654_at Dirc2 0.48 1443621_at Xaf1 1.92 1456796_at Snai30.48 1425078_x_at C130026I21Rik 1.92 1417702_a_at Hnmt 0.48 1427994_atCd300lf 1.91 1450738_at Kif2 la 0.48 1456545_at Il18rap 1.91 1422007_atAqp3 0.48 1418346_at Insl6 1.91 1417014_at Hspb8 0.47 1428372_at St51.91 1438422_at Lrrc20 0.47 1417045_at Bid 1.91 1417029_a_at Trim2 0.471433699_at Tnfaip3 1.91 1417673_at Grb14 0.47 1440275_at Runx3 1.911428471_at Sorbs1 0.47 1460253_at Cmtm7 1.91 1439117_at Clmn 0.471421188_at Ccr2 1.90 1427946_s_at Dpyd 0.46 1427911_at Tmem173 1.901418317_at Lhx2 0.46 1420653_at Tgfb1 1.90 1424367_a_at Homer2 0.461437643_at Cenpj 1.90 1454691_at Nrxn1 0.46 1443687_x_at H2-DMb2 1.901419082_at Serpinb2 0.46 1440299_at E330016A19Rik 1.90 1434651_a_atCldn3 0.46 1425151_a_at Noxo1 1.90 1460569_x_at Cldn3 0.45 1441097_atVangl1 1.90 1431056_a_at Lpl 0.45 1449988_at Gimap1 1.89 1443832_s_atSdpr 0.45 1427404_x_at Gm5506 1.89 1451701_x_at Cldn3 0.45 1425617_atDhx9 1.88 1419490_at Fam19a5 0.45 1426926_at Plcg2 1.88 1428792_at Bcas10.45 1424037_at Itpka 1.88 1419492_s_at Defb1 0.45 1449925_at Cxcr3 1.881424393_s_at Adhfe1 0.44 1435394_s_at Rhoc 1.87 1448978_at Ngef 0.441453392_at Ttc39b 1.87 1429344_at 9.13E+15 0.44 1449473_s_at Cd40 1.871437637_at Phtf2 0.44 1419186_a_at St8sia4 1.86 1425826_a_at Sorbs1 0.441455898_x_at Slc2a3 1.86 1451620_at C1ql3 0.44 1430447_a_at Lair1 1.861450759_at Bmp6 0.43 1418353_at Cd5 1.85 1433638_s_at Hoxd8 0.431449425_at Wnt2 1.85 1415996_at Txnip 0.43 1439114_at Ddx60 1.851448551_a_at Trim2 0.43 1425084_at Gimap7 1.85 1426981_at Pcsk6 0.431425819_at Zbtb7c 1.85 1427673_a_at Sema3e 0.43 1416001_a_at Cotl1 1.841451527_at Pcolce2 0.43 1422704_at Gyk 1.84 1434877_at Nptx1 0.421445452_at Traf1 1.84 1453435_a_at Fmo2 0.42 1438852_x_at Mcm6 1.841449621_s_at Thsd1 0.42 1436423_at Themis 1.84 1431805_a_at Rhpn2 0.421417599_at Cd276 1.84 1449824_at Prg4 0.41 1421930_at Icos 1.841416129_at Errfi1 0.41 1421814_at Msn 1.83 1434628_a_at Rhpn2 0.411419132_at Tlr2 1.83 1455214_at Mitf 0.41 1448568_a_at Sklc20a1 1.831427345_a_at Sult1a1 0.41 1423602_at Traf1 1.83 1418205_at Thsd1 0.401451931_x_at H2-L 1.82 1419414_at Gng13 0.40 1456836_at Itk 1.821437840_s_at D2hgdh 0.40 1453129_a_at Rgs12 1.82 1423635_at Bmp2 0.401426454_at Arhgdib 1.82 1423679_at 2810432L12Rik 0.39 1451385_at Fam162a1.82 1418063_at Kera 0.39 1419470_at Gnb4 1.82 1434667_at Col8a2 0.391435697_a_at Cytip 1.82 1424649_a_at Tspan8 0.39 1424214_at Parm1 1.811446498_at Il20ra 0.39 1458524_at Fndc3a 1.81 1441918_x_at Serpinb6e0.39 1455051_at Rnf31 1.81 1415997_at Txnip 0.39 1452163_at Ets1 1.811431833_a_at Hmgcs2 0.38 1448328_at Sh3bp2 1.80 1418589_a_at Mlf1 0.381456700_x_at Marcks 1.80 1419717_at Sema3e 0.38 1451784_x_at H2-D1 1.801429166_s_at Clmn 0.38 1437606_at Btbd19 1.80 1435459_at Fmo2 0.381439476_at Dsg2 1.80 1434669_at Ralgps1 0.38 1440878_at Runx1 1.791431099_at Hoxd8 0.38 1453472_a_at Slamf7 1.79 1434657_at Gls 0.371434653_at Ptk2b 1.79 1418595_at Plin4 0.36 1436708_x_at Mcm4 1.791434474_at Abca5 0.36 1454592_at 9430012M22Rik 1.79 1417168_a_at Usp20.36 1433954_at 4632419I22Rik 1.78 1427029_at Htra3 0.35 1426315_a_at6330416G13Rik 1.78 1456895_at Cd209b 0.34 1451289_at Dclk1 1.781416225_at Adh1 0.34 1443686_at H2-DMb2 1.78 1454969_at Lypd6 0.341449903_at Crtam 1.78 1456741_s_at Gpm6a 0.34 1418090_at Plvap 1.781442226_at Sema3e 0.33 1460250_at Sostdc1 1.77 1451478_at Angptl7 0.331435749_at Gda 1.77 1418706_at Slc38a3 0.32 1450269_a_at Pfkl 1.771436279_at Slc26a7 0.32 1438910_a_at Stom 1.77 1460244_at Upb1 0.321437868_at Fam46a 1.77 1459737_s_at Ttr 0.31 1448449_at Ripk3 1.771419816_s_at Errfi1 0.31 1424496_at 5133401N09Rik 1.77 1417765_a_at Amy10.30 1438854_x_at Pitpnm1 1.77 1446681_at BB086117 0.30 1424930_s_atFam83f 1.77 1433837_at 8430408G22Rik 0.30 1460378_a_at Tes 1.771454965_at Fam171b 0.29 1448914_a_at Csf1 1.76 1433596_at Dnajc6 0.291448659_at Casp7 1.76 1425357_a_at Grem1 0.29 1427689_a_at Tnip1 1.761455913_x_at Ttr 0.29 1428242_at Hmha1 1.76 1433836_a_at 8430408G22Rik0.28 1451382_at Chac1 0.28 1454608_x_at Ttr 0.26 1433877_at Fam46b 0.261425281_a_at Tsc22d3 0.25 1451535_at Il31ra 0.24 1423405_at Timp4 0.231435184_at Npr3 0.23 1437665_at Il22ra2 0.22 1456512_at Pdzrn4 0.221417788_at Sncg 0.21 1460012_at Wfdc3 0.20 1452543_a_at Scgb1a1 0.191419332_at Egfl6 0.19 1436643_x_at Hamp2 0.18 1453327_at Krt24 0.171456487_at Adcy1 0.14 1449545_at Fgf18 0.12

By comparing the corresponding lists of genes identified asdifferentially expressed to each other (FIG. 55A, bottom panel), threestriking gene expression signatures were uncovered in this analysis.First, many of the upregulated genes in alopecic skin of both specieswere IFN-response genes, including the IFN-inducible chemokines CXCL9,10 and 11¹¹ (FIG. 55A, FIG. 60). Secondly, several key CTL-specifictranscripts were identified, including CD8A and granzymes A and B (FIG.55A).

In turn, using CTL effectors, the small molecule JAK3 inhibitor,tofacitinib (JAK3>JAK1>>JAK2 selectivity)¹⁸, was shown to block IL-15triggered pSTAT3 activation (FIG. 55B), and inhibit CTL effectorfunctions, including dermal sheath cell cytotoxicity (FIG. 55C), andgranzyme B/IFNγ production (FIG. 55D). Thus, hair follicle production ofIL-15 provides a cytokine axis that can be interrupted pharmacologicallyby inhibiting its signaling in immune effectors.

Next, it was determined whether the effects of IL-15 blockade could berecapitulated by intervening downstream using small molecule inhibitorsof JAK3. Using tofacitinib, systemic administration of JAK3i (FIG. 56)treatment prevented the development of AA and the expansion ofCD8⁺NKG2D⁺ T cells in all grafted recipients. Skin of mice treated withthe protein tyrosine kinase inhibitor (PTKi) showed no histologicalsigns of inflammation and prevented the emergence of the skin ALADIN(Alopecia Areata Disease Activity Index) transcriptional signature andGene Expression Dynamic Index (GEDI) analysis in all mice (FIG. 56).

To evaluate the efficacy of JAK3i in the clinical context of AA, it wasdetermined whether systemic JAK3i treatment could reverse establisheddisease by initiating therapy 7 weeks after grafting, a time point atwhich all mice had developed extensive AA. Importantly, systemic therapyinduced hair regrowth all over the body, and likewise eliminated theexpansion of CD8⁺NKG2D⁺ T cells and reversed histological markers ofdisease (FIG. 61), an effect that persisted 2-3 months after thecessation of treatment.

Finally, to test a more clinically convenient route of delivery, it wasdetermined whether topical administration of PTKis could reverselong-standing alopecia areata with similar kinetics as systemicdelivery. In established disease, topical tofacitinib, was highlyeffective in reversing disease in treated lesions (applied to backskin), and complete hair regrowth was observed within 12 weeks followingtopical therapy (FIG. 57A-B). Topical therapy was associated with thedisappearance of CD8⁺NKG2D⁺ T cells in the treated skin (FIG. 57C) andthe reversal of histological markers of disease (FIG. 57D), as well asthe normalization of the ALADIN transcriptional signature (FIG. 57E),and the GEDI index in all treated mice (FIG. 57F). Notably, untreatedareas on the abdomen remained alopecic (FIG. 57A, middle panels),demonstrating that topical therapy was locally effective and therapeuticeffects were not the result of systemic absorption. Further, theseeffects were durable 2-3 months after the cessation of treatment (FIG.57A, right panels).

Taken together, the data identify CD8⁺NKG2D⁺ T cells as the immunecellular effectors responsible for autoimmune attack of the hairfollicle and provide support for a model of AA pathogenesis.

Importantly, these common mechanistic underpinnings were first revealedby in the GWAS study, which placed AA squarely among this group ofallied autoimmune diseases involving NKG2DL mediated recruitment of CD8T cell effectors, including type I diabetes^(23,24), celiacdisease^(16,25) and rheumatoid arthritis²⁶. These pathways can beinterrupted by small molecule inhibitors of the IL-15 downstreameffector JAK kinases, the latter being particularly appealing as atopical therapeutic approach in this cutaneous disease. FDA-approved JAKinhibitors were used to show the therapeutic effect, arguing forclinical evaluation in AA with these compounds or other JAK PTKis inclinical development²⁸.

In a very short time since the GWAS findings, not only have the specificsubset of cytotoxic T cells that give rise to AA been identified, butthey have successfully targeted them for elimination using clinicallyrelevant, rational therapies selected on the basis of our mechanisticstudies. The findings illustrate the power of GWAS studies in uncoveringnew disease pathways, and, coupled with translational approaches²⁹, haverapidly opened a new and unexpected avenue for intervention in AA.

Methods

Mice.

7-10 week old female C57B1.6 and C3H/HeJ mice (Jackson Laboratories, BarHarbor, Me.) were used and maintained under specific pathogen-freeconditions.

Transfer of Alopecia Areata Using Grafted C3H/HeJ Skin.

Normal-haired C3H/HeJ mice were grafted at 8 weeks of age (during thesecond telogen) with skin from a C3H/HeJ mouse that developed AAspontaneously, as described previously-. In brief, mice spontaneouslyaffected with AA were euthanized, full thickness skin grafts ofapproximately 2 cm in diameter were removed and grafted to normal-hairedC3H/HeJ mice. Hair loss typically began at around 4-6 weeks aftergrafting.

Flow Cytometric Analysis of Skin and Cutaneous Lymph Nodes.

To make a single cell suspension of mouse skin, fat was removed from theoverlying skin in cold PBS and then incubated in collagenase type I (2mg/ml in PBS) at 32° C. for 75 minutes. After digestion the skin wasminced in RPMI/10% fetal bovine serum, filtered through a 70 mM cellstrainer, and centrifuged at 1100 g for 5 minutes. The pellet wasresuspended in RPMI/10% fetal bovine serum, filtered through a 40 mMcell strainer and spun at 400 g for 5 mins. The pellet was resuspendedin FACs buffer (PBS/5% BSA), DAPI to gate on live cells and stainingantibodies (listed in Supplemental Data). Cutaneous lymph nodes werepooled minced in RPMI, filtered through a 40 mM cell strainer,centrifuged at 400 g for 5 minutes stained and analysed on a BD LSR IIflow cytometer.

Transfer of T Cell Populations into Recipient C3H/HeJ Mice.

For positive selection of T cell populations, lymph node cells wereobtained from 5 C3H/HeJ alopecic mice, stained with anti-CD4, anti-CD8,and anti-NKG2D antibodies, then sorted (BD Influx) to obtain twofractions: CD8⁺NKG2D⁺ T cells, and CD8⁺NKG2D⁻ T cells. Three to five7-week-old C3H/HeJ mice per group were injected subcutaneously with twomillion sorted cells of each population. For negatively selectedpopulations, NKG2D⁺ cells were depleted by incubating total lymph nodecells from 3 alopecic C3H/HeJ mice with biotinylated anti-NKG2D (CX5)and then with streptavidin-conjugated beads (Miltenyi) prior to removalon a Miltenyi magnetic column. Five million cells (eitherCD8/NKG2D-depleted or total lymph node cells) were suspended in 100 ulPBS and transferred into each of 5 mice by subcutaneous injection.

Prevention and Treatment Studies.

For prevention studies, mice were treatment beginning the day ofgrafting (n=5-10 mice per group). For JAK3i experiments: mice wereimplanted subcutaneously with ALZET™ osmotic mini-pumps (pumps, model2004, Durect Corporation) on the back of each mouse to deliver vehicle(poly(ethylene glycol) (PEG)300) or vehicle containing the JAK3itofacitinib (Abmole) at 15 mg/kg/day for 12 weeks.

For topical treatment studies, grafted mice with long-standing AA (morethan 8 weeks) were treated once daily for 12 weeks to affected skin onthe dorsal back with vehicle (AQUAPHOR™) or vehicle containing the JAK3inhibitor (0.5% ointment). Full-thickness skin biopsies were excisedfrom the dorsal surface of each mouse at interim time points, and skinsamples were either snap frozen in liquid nitrogen for RNA extraction orsnap frozen in OCT for immunostaining. Hair status was examined twiceweekly and hair growth index calculated as described.

Immunohistochemistry and Immunofluorescence.

8 mM acetone-fixed frozen mouse skin sections were air-dried and stainedovernight with anti-mouse Abs (see below) at 4° C. in a moist chamber.Human hair follicles were microdissected and embedded in OCT compoundprior to sectioning and staining (see below).

Primary Dermal Sheath and Lymphokine-Activated Killer (LAK) CellCulture.

Dermal sheath (DS) cells were isolated from microdissected mousevibrissa follicles and cultured in 20% FBS DMEM with 5 ng/ml murine FGF(Pepro Tech). LAK cells were generated from splenocytes plated at 4×10⁶in 6-well plates with 50 nM JAK3i (tofacitinib) or 50 ng/ml murine IL-15plus 50 nM JAK3i, and incubated at 37° C. in a 5% CO₂ incubator for 96hours.

In Vitro Cytotoxicity Assays.

Determination of specific killing of target cells was performed usingCFSE-labeled DS cells at targets mixed with different ratios of effectorcells incubated for 5 hours at 37° C. 5% CO2 with or withoutneutralizing rat anti-mouse NKG2D antibody (20 ug/ml) (Biolegend, CX5).Specific lysis of DS cells was determined flow cytometrically bymeasuring cell death of CFSE+ DS cells using Annexin V/7-AAD.

Gene Expression Analysis in Human and Mouse Skin and T Cells.

Total RNA was isolated using the MIRNEASY™ Mini Kit (Qiagen Inc.,Valencia, Calif., USA) with on-column DNA digestion using the RNase-freeDNase set (Qiagen, Inc.). For RNAseq analysis CD3+CD8+CD44+NKG2D+ andCD3+CD8+CD44+NKG2D− cells were flow-sorted from lymph nodes of alopecicC3H/HeJ mice. RNA was extracted as above and prepared for RNA-seq usingthe TRUSEQ™ RNA Sample Prep Kit v2. Samples were sequenced on the HISEQ™2000 sequencer (Illumina, San Diego, Calif.) for 50 cycles. RNA-Seqfiles were demultiplexed by the Rockefeller University Genomics CoreFacility.

For global transcriptional profiling in mouse skin, total extracted RNAwas processed using the 3′ IVT EXPRESS KIT™ from AFFYMETRIX™. Resultingbiotinylated cDNA samples were hybridized to the Mouse Genome 430 2.0gene chips and subsequently washed, stained withstreptavidin-phycoerythrin, and scanned on an HP GENEARRAY™ Scanner(Hewlett-Packard Company, Palo Alto, Calif.).

For human AA samples, perilesional punch biopsies from 5 patients withpatchy alopecia areata who were not undergoing local or systemictreatments were collected and compared to scalp biopsies from 5unrelated unaffected individuals.

Extracted total RNA were reverse transcribed and amplified using theOVATION™ RNA Amplification V2 kit (NuGEN Technologies, Inc., San Carlos,Calif.). Amplified cDNA was biotinylated with the ENCORE™ Biotin Module(NuGEN Technologies) and then hybridized to the U133A Plus 2.0 genechips.

Antibodies Used for Flow Cytometry and Immunostaining.

Flow cytometric analysis used the following anti-mouse antibodies: CD3(17A2, Ebioscience), CD4 (GK1.5, BD), CD8a (53-6.7, BD), CD8a(YTS156.7.7, Biolegend), NKG2D (CX5, Ebioscience), NKG2A/C/E (clone20d5, Ebioscience), CD44 (IM7, BD), CD45 (30-F11, BD), CD49b (Dx5, BD),CD62L (MEL-14, BD), CD69 (H1.2F3, BD), CD103 (2E7, eBioscience), IFNγ(XMG1.2, Ebioscience), Granzyme B (NGZB, eBioscience), Rae-1 (186107,R&D).

For immunohistochemical studies of mouse skin, 8 μM methanol-fixedfrozen skin sections were stained with primary rat antibodies(Biolegend) including: anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7),Biotin anti-MHC class I (clone 36-7.5), anti-MHC class II (cloneM5/114.15.2). Biotinylated goat anti-rat IgG (Life Technologies) wasused as secondary antibody. For immunofluorescence studies anti-H60(R&D, clone 205326), anti-Pan Rae-1(R&D, clone 186107), anti-NKG2D (R&Dclone 191004), anti-K71 (Abcam) primary antibody were used inimmunofluorescence. ALEXA FLUOR™ 488 or ALEXA FLUOR™ 594-conjugated goatanti-Rat, donkey anti-Rabbit or donkey anti-Goat antibody was used assecondary antibody (Life Technologies).

Human hair follicles were microdissected and embedded in OCT compoundprior to sectioning and staining. 8 μM methanol-fixed frozen sectionswere stained with CD8 (SCBT, C8/144B) followed by staining with AlexaFluor 488 or Alexa Fluor 594-conjugated secondary antibody (LifeTechnologies). All images were captured with an SDRC Zeiss ExciterConfocal Microscope.

Statistical Analysis and Quality Control of Gene Expression Signatures

RNA-Seq Analysis.

Samples were sequenced on the HiSeq 2000 sequencer (Illumina, San Diego,Calif.) for 50 cycles. RNA-Seq files were demultiplexed by theRockefeller University Genomics Core Facility. Quality control of thesample fastq files was performed using fastqc^(S1). TopHat,^(S2) wasused to map transcripts to the UCSC mm9 reference genome from iGenome.The RefSeq gene annotation packaged with this iGenome version of theUCSC mm9 were used. The htseq-count utility from the HTSeq package wasused to convert TopHat bam files to counts that could be used as inputfor downstream analysis of differential expression with edgeR^(S3).Absent genes were removed and a pseudocount of 1 was added in order toavoid division by zero in downstream analysis. EdgeR was used toidentify differentially expressed genes using a matched pairs designwith three biological replicates.

Microarray Analysis.

Quality Control, Preprocessing.

For the mouse cDNA samples were hybridized to the Mouse Genome 430 2.0gene chips and subsequently washed, stained withstreptavidin-phycoerythrin, and scanned on an HP GeneArray Scanner(Hewlett-Packard Company, Palo Alto, Calif.). For the human, amplifiedcDNA was hybridized to the U133A 2.0 gene chips.

Quality control was performed using the affyanalysisQC package fromarravanalysis.org/. AffyanalysisQC uses R/BioConductor packages: affy,affycomp, affypdnn, affyPLM, affyQCReport, ArrayTools, bioDistm biomaRt,simpleaffy, yaqcaffy to perform QC within a single script. RMAnormalization.^(S4) was performed on each experimental group separately.Batch effect correction using ComBat was required for the preventionexperiments.

Microarray preprocessing was performed using BioConductor in R.Preprocessing of the three experiments, 1) spontaneous AA mice vs.normal mice, 2) prevention mice with three treatments vs placebo andsham-operated mice, and 3) treatment mice for two treatments vs. placebowere performed separately using the same pipeline. In addition to thepreprocessing that was done for the mouse skin samples, Harshlight wasused to correct for image defects for the human skin samples.

Identification of Gene Signatures.

Unsupervised Analysis.

Hierarchical clustering was performed using Cluster′^(S5) on the 363genes from the human 5×5 and 583 genes from the spontaneous mouse 3×3 inorder that met the threshold abs(log FC)>1, unadjusted p-value<=0.05.Genes were first selected that met the threshold log FC>1, andunadjusted p-value<=0.05. Genes were median centered and normalized.Spearman rank correlation was used as the similarity measure and averagelinkage was used to perform row (genes) and column (sample) clustering.Visualization of the hierarchical clusters was performed with javaTreeView.^(S6). Gene Expression Dynamic Index (GEDI) analysis was usedto visualize how “metagenes” identified with a self organizing mapalgorithm vary across samples^(S7). Metagenes are clusters of genes thatshow similar expression patterns across samples and that are assigned toa single pixel in a two dimensional grid. Neighboring pixels demonstratesimilar expression patterns to one another.

Supervised Analysis.

Initial analysis of differential gene expression was performed on thespontaneous mouse 3×3 and the human 5×5 data sets using limma′^(S8). Athreshold of 1.5 fold change and unadjusted p-value of 0.05.

Rt-PCR Validation.

Predicted differentially expressed genes in human and mouse wereconfirmed using RT-PCR. First-strand cDNA was synthesized using a ratioof 2:1 random primers: Oligo (dT) primer and SUPERSCRIPT™ III RT(Invitrogen) according to the manufacturer's instructions. qRT-PCR wasperformed on an ABI 7300™ machine and analyzed with ABI RelativeQuantification Study™ software (Applied Biosystems, Foster City, Calif.,USA). Primers were designed according to ABI guidelines and allreactions were performed using Power SYBR™ Green PCR Master Mix (AppliedBiosystems), 250 nM primers (Invitrogen) and 20 ng cDNA in a 20 μLreaction volume. Primer sequences are provided in Table 16.

TABLE 16 RT-PCR Primers for mouse mRNA validation studies. Mouse RT-PCRSEQ ID SEQ ID Gene Forward Primer NO: Reverse Primer NO: Cd8aGAGACCAGAAGAT 12 GCCTGGGACATTTGC 13 TGTCGGC AAACA Cd3d ACTGTGTGGAGCT 14CTGTACTGGGTATCTT 15 AGACTCG CACG Stat1 CGCAACTACAAAG 16 ATCCAGTTCGCTTAG17 TCATGGC GGTCG Ifng ACGGCACAGTCAT 18 GCTGATGGCCTGATT 19 TGAAAGC GTCTTTap1 CCTGCTTATCTTGG 20 GGTAGCACCCTCCTCT 21 ATGATGC CTT Cxcl9ACGGAGATCAAAC 22 TTCCCCCTCTTTTGCT 23 CTGCCTA TTTT Cxcl10 ATCCACCGCTGAG24 CCTTGAGTCCCACTCA 25 AGACATC GACC Cxcl11 TAGCCCTGGCTGC 26ACTTTGTCGCAGCCGT 27 AATATCT TACT Irf7 GCACCCTCCTTTTC 28 GCCAAGGTGGCTGTA29 ACTGAG GATGT Ccl5 CCCTCACCATCAT 30 GAGCACTTGCTGCTG 31 CCTCACT GTGTAH60a TGCCTGATTCTGA 32 ATTCACTGAGCACTG 33 GCCTTTTCA TCCATGTAGAT H60bAGGCCTTTTGGTC 34 ATGTTTTTTATCACCA 35 CTGCTGAAT AAATCAAGGAGT H60cAGATTTCAGTTGC 36 ACATGTGCAGCAGTG 37 TGCCTCA GTTG *Commercial Solarisreal-time PCR system primers and probe sets AX-047904-01, AAX-047854-01,AX-047833-01, AX-049954-01, AX-042357-01 and AX-040917-00 (ThermoScientific) were used for PCR of Rae-1a, Rae-1b, Rae-1c, Rae-1d, Rae-1eand GAPDH. Results are expressed as ‘fold increase’ in mRNA expression.

TABLE 17 RT-PCR Primers for human mRNA validation studies. Human RT-PCRSEQ SEQ ID ID Gene NO. Forward Primer NO: Reverse Primer GZMA 38AGATTTCTGGCATCCTCTC 39 GACCATGTAGGGTCTTGA IL15 40 TTTCAGTGCAGGGCTTCCT 41GGGTGAACATCACTTTCCC STAT1 42 GCAGGTTCACCAGCTTTAT 43 TGAAGATTACGCTTGCTTTCXCL9 44 GTAGTGAGAAAGGGTCGC 45 AGGGCTTGGGGCAAATTG IFNG 46TCGGTAACTGACTTGAATGTCCA 47 TCGCTTCCCTGTTTTAGCTGC

The following PCR protocol was used: step 1: 50° C. for 2 min; step 2:95° C. for 10 min; step 3: 95° C. for 15 s; step 4: 60° C. for 1 min;repeat steps 3 and 4 for 40 cycles. All samples were run inquadruplicate for three independent runs and normalized against anendogenous internal control as indicated.

ALADIN Scores.

The IFN and CTL signatures were used to develop a bivariate scorestatistic. Individual signature IFN and CTL scores were determinedfollowing procedures used in human SLE^(S9,S10). The sets of genesselected to comprise our IFN and CTL signatures were CD8A, GZMB, andICOS for the CTL signature, and CXCL9, CXCL10, CXCL11, STAT1, and MX1for the IFN signature. The scores for the prevention mice werecalculated in relation to the sham mice; whereas, the scores for thetopical treatment experiments were calculated relative to all thesamples at week zero.

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Example 11—Effect of JAK3 Inhibitor on the Induction of Hair Growth

The JAK-STAT signaling pathway has been implicated in severaldevelopmental processes, and most recently in stem cell maintenance,activation and differentiation. In the course of the inventor's topicaltreatment studies using JAK3 in the C3H/HeJ AA mouse model, it wasnoticed that the hairs that regrew did so with two striking featuresthat were different from systemic administration: 1) hair regrowth wasvery rapid; and 2) the hair coat was darkly pigmented. Without beingbound by theory, in addition to the JAK3 inhibitor eliminatingpathogenic T cells from the skin, the JAK3 inhibitor also has a directeffect on the hair follicle itself, for example, via an anagen-promotingeffect.

The dynamics of the JAK-STAT signaling pathway were first interrogatedusing a targeted RT-PCR array containing readouts of JAK-STAT signaling(FIG. 63), and compared gene expression between the telogen vs. anagenstages of the normal hair cycle (FIGS. 64-66).

This data revealed that many components of JAK-STAT signaling wereupregulated in telogen and downregulated in anagen phase of the normalhair cycle, indicating that in the context of the hair cycle, JAK-STATsignaling can be associated with maintaining stem cell quiescence intelogen (FIG. 66).

To test whether inhibition of JAK-STAT signaling could therefore triggerthe telogen-to-anagen transition, a topical JAK3 inhibitor was appliedto test whether anagen could be induced in normal mouse skin in telogen.

Indeed, topical administration of a JAK3 inhibitor resulted in astriking anagen induction in mouse skin in telogen, compared to vehiclealone. This was associated with marked proliferation of keratinocytematrix cells, and the induction and growth of robust pigmented anagenhairs after 1-2 weeks (see FIGS. 69-74).

This observation was compared to a positive control SAG (sonic hedgehogagonist) which is known to have the same effect, and the JAK3 inhibitorwas comparable in its effect on anagen induction (FIGS. 69, 73, 74).

Without being bound by theory, these findings indicate that blockade ofJAK-STAT signaling in telogen mimics in part the molecular events ofanagen initiation, and can be a useful therapeutic agent for hairgrowth, using topical JAK inhibitors to induce telogen hairs to re-enteranagen.

What is claimed:
 1. A method of treating a hair loss disorder in asubject in need thereof, the method consisting of: administering to thesubject an effective amount of a small molecule janus kinase (JAK) 1and/or JAK2 inhibitor, wherein the JAK1 and/or JAK2 inhibitor comprisesone or more isotopes.
 2. The method of claim 1, wherein the JAK1 and/orJAK2 inhibitor is selected from ruxolitinib (INCB018424), filgotinib(GLPG0634), Tyrphostin B42 (AG490), momelotinib (CYT387), pacritinib(SB1518), baricitinib (LY3009104), AZD1480, fedratinib (TG101348),BMS-911543, and lestaurtinib (CEP-701), a pharmaceutically acceptablesalt thereof, or a combination thereof.
 3. The method of claim 1,wherein the hair loss disorder is androgenetic alopecia, telogeneffluvium, tinea capitis, patchy alopecia areata, alopecia totalis,hypotrichosis, hereditary hypotrichosis simplex, alopecia universalis,or a combination thereof.
 4. The method of claim 1, wherein the hairloss disorder is alopecia areata.
 5. The method of claim 1, wherein thehair loss disorder is patchy alopecia areata.
 6. The method of claim 1,wherein the hair loss disorder is alopecia totalis.
 7. The method ofclaim 1, wherein the hair loss disorder is alopecia universalis.
 8. Themethod of claim 1, wherein the hair loss disorder is androgeneticalopecia.
 9. The method of claim 1, wherein the JAK1 and/or JAK2inhibitor is administered topically.
 10. The method of claim 1, whereinthe JAK1 and/or JAK2 inhibitor is administered orally.
 11. The method ofclaim 1, wherein the JAK1 and/or JAK2 inhibitor is administered via aroute selected from the group consisting of intra-muscular injection,intra-peritoneal injection, an infusion, intravenous, intradermal,subcutaneous, oral, inhalation, transdermal, topical, transmucosal,nasal, rectal, and a combination thereof.
 12. The method of claim 1,wherein the JAK1 and/or JAK2 inhibitor is administered in apharmaceutical composition selected from the group consisting of asolution, suspension, capsule, tablet, pill, troche, nasal spray,suppository, ointment, salve, gel, or cream.
 13. The method of claim 1,wherein administering to the human occurs twice daily, daily, weekly,twice weekly, monthly, twice monthly, yearly, 1 time per week, 2 timesper week, 3 times per week, 4 times per week, 5 times per week, 6 timesper week, 7 times per week, 8 times per week, 9 times per week, 10 timesper week, 11 times per week, 12 times per week, 13 times per week, or 14times per week.
 14. The method of claim 1, wherein treating a hair lossdisorder is by inducing hair growth.
 15. The method of claim 1, whereinthe compound is suitable for nuclear magnetic resonance (NMR).
 16. Themethod of claim 1, wherein the one or more isotopes is a radioisotope.17. The method of claim 1, wherein the one or more isotopes are isotopesof carbon or hydrogen.
 18. The method of claim 17, wherein the hair lossdisorder is patchy alopecia areata.
 19. The method of claim 17, whereinthe hair loss disorder is alopecia totalis.
 20. The method of claim 17,wherein the hair loss disorder is alopecia universalis.
 21. The methodof claim 17, wherein the JAK1 and/or JAK2 inhibitor is administeredtopically.
 22. The method of claim 17, wherein JAK1 and/or JAK2inhibitor is administered orally.
 23. The method of claim 17, whereinthe JAK1 and/or JAK2 inhibitor is suitable for NMR.
 24. The method ofclaim 1, wherein the effective amount of the JAK1 and/or JAK2 inhibitoradministered to the subject is selected from about 0.0001 μg/kg bodyweight, about 0.00025 μg/kg body weight, about 0.0005 μg/kg body weight,about 0.00075 μg/kg body weight, about 0.001 μg/kg body weight, about0.0025 μg/kg body weight, about 0.005 μg/kg body weight, about 0.0075μg/kg body weight, about 0.01 μg/kg body weight, about 0.025 μg/kg bodyweight, about 0.05 μg/kg body weight, about 0.075 μg/kg body weight,about 0.1 μg/kg body weight, about 0.25 μg/kg body weight, about 0.5μg/kg body weight, about 0.75 μg/kg body weight, about 1 μg/kg bodyweight, about 5 μg/kg body weight, about 10 μg/kg body weight, about 25μg/kg body weight, about 50 μg/kg body weight, about 75 μg/kg bodyweight, about 100 μg/kg body weight, about 150 μg/kg body weight, about200 μg/kg body weight, about 250 μg/kg body weight, about 300 μg/kg bodyweight, about 350 μg/kg body weight, about 400 μg/kg body weight, about450 μg/kg body weight, about 500 μg/kg body weight, about 550 μg/kg bodyweight, about 600 μg/kg body weight, about 650 μg/kg body weight, about700 μg/kg body weight, about 750 μg/kg body weight, about 800 μg/kg bodyweight, about 850 μg/kg body weight, about 900 μg/kg body weight, about950 μg/kg body weight, about 1000 μg/kg body weight, about 2000 μg/kgbody weight, about 3000 μg/kg body weight, about 4000 μg/kg body weight,about 5000 μg/kg body weight, about 6000 μg/kg body weight, about 7000μg/kg body weight, about 8000 μg/kg body weight, about 95000 μg/kg bodyweight, or about 10,000 μg/kg body weight, about 1 mg/kg body weight,about 1.5 mg/kg body weight, about 2 mg/kg body weight, about 2.5 mg/kgbody weight, about 3 mg/kg body weight, about 3.5 mg/kg body weight,about 4 mg/kg body weight, about 4.5 mg/kg body weight, about 5 mg/kgbody weight, about 5.5 mg/kg body weight, about 6 mg/kg body weight,about 6.5 mg/kg body weight, about 7 mg/kg body weight, about 7.5 mg/kgbody weight, about 8 mg/kg body weight, about 9.5 mg/kg body weight,about 10 mg/kg body weight, about 10.5 mg/kg body weight, about 11.0mg/kg body weight, about 11.5 mg/kg body weight, about 12 mg/kg bodyweight, about 12.5 mg/kg body weight, about 13 mg/kg body weight, about13.5 mg/kg body weight, about 14 mg/kg body weight, about 14.5 mg/kgbody weight, about 15 mg/kg body weight, about 15.5 mg/kg body weight,about 16 mg/kg body weight, about 16.5 mg/kg body weight, about 17 mg/kgbody weight, about 17.5 mg/kg body weight, about 18 mg/kg body weight,about 19.5 mg/kg body weight, about 20 mg/kg body weight, about 21.5mg/kg body weight, about 22 mg/kg body weight, about 22.5 mg/kg bodyweight, about 23 mg/kg body weight, about 23.5 mg/kg body weight, about24 mg/kg body weight, about 24.5 mg/kg body weight, about 25 mg/kg bodyweight, about 25.5 mg/kg body weight, about 26 mg/kg body weight, about26.5 mg/kg body weight, about 27 mg/kg body weight, about 27.5 mg/kgbody weight, about 28 mg/kg body weight, about 29.5 mg/kg body weight,or about 30 mg/kg body weight.
 25. The method of claim 1, whereinadministering to the subject occurs for at least 1 week.